Mirac proteins

ABSTRACT

This disclosure relates to a method of generating conditionally active biologic proteins from wild type proteins, in particular therapeutic proteins, which are reversibly or irreversibly inactivated at the wild type normal physiological conditions. For example, evolved proteins are virtually inactive at body temperature, but are active at lower temperatures.

RELATED APPLICATION INFORMATION

This application is a divisional of U.S. patent application Ser. No.13/523,509 filed Jun. 12, 2012 which is a continuation of U.S. patentapplication Ser. No. 13/255,676 filed on Sep. 9, 2011, which is a 371continuation of international application no. PCT/US2010/026611 filed onMar. 9, 2010, designating the United States of America, now expired,which is a nonprovisional of U.S. provisional application No. 61/209,489filed on Mar. 9, 2009, now expired.

FIELD OF THE DISCLOSURE

This disclosure relates to the field of protein evolution and activity.Specifically, this disclosure relates to a method of generatingconditionally active biologic proteins from wild type proteins, inparticular therapeutic proteins, and which are reversibly orirreversibly inactivated at the wild type normal physiologicalconditions. For example, evolved proteins are virtually inactive at bodytemperature, but are active at lower temperatures.

BACKGROUND OF THE DISCLOSURE

There is a considerable body of literature describing the potential forevolving proteins for a variety of characteristics, especially enzymesfor example, to be stabilized for operation at different conditions. Forexample, enzymes have been evolved to be stabilized at highertemperatures, with varying activity. In situations where there is anactivity improvement at the high temperature, a substantial portion ofthe improvement can be attributed to the higher kinetic activitycommonly described by the Q10 rule where it is estimated that in thecase of an enzyme the turnover doubles for every increase of 10 degreesCelsius. In addition, there exist examples of natural mutations thatdestabilize proteins at their normal operating conditions, such aswild-type temperature activity of the molecule. For temperature mutants,these mutants can be active at the lower temperature, but typically areactive at a reduced level compared to the wild type molecules (alsotypically described by a reduction in activity guided by the Q10 orsimilar rules).

It is desirable to generate useful molecules that are conditionallyactivated, for example virtually inactive at wild-type conditions butare active at other than wild-type conditions at a level that is equalor better than at wild-type conditions, or that are activated orinactivated in certain microenvironments, or that are activated orinactivated over time. Besides temperature, other conditions for whichthe proteins can be evolved or optimized include pH, osmotic pressure,osmolality, oxidation and electrolyte concentration. Other desirableproperties that can be optimized during evolution include chemicalresistance, and proteolytic resistance.

Many strategies for evolving or engineering molecules have beenpublished. However, engineering or evolving a protein to be inactive orvirtually inactive (less than 10% activity and especially 1% activity)at its wild type operating condition, while maintaining activityequivalent or better than its wild type condition at new conditions,requires that the destabilizing mutation(s) co-exist with activityincreasing mutations that do not counter the destabilizing effect. It isexpected that destabilization would reduce the protein's activitygreater than the effects predicted by standard rules such as Q10,therefore the ability to evolve proteins that work efficiently at lowertemperature, for example, while being inactivated under their normaloperating condition, creates an unexpected new class of proteins werefer to as Mirac Proteins.

Throughout this application, various publications are referenced byauthor and date. The disclosures of these publications in theirentireties are hereby incorporated by reference into this application inorder to more fully describe the state of the art as known to thoseskilled therein as of the date of the disclosure described and claimedherein.

SUMMARY OF THE DISCLOSURE

The disclosure provides a method of preparing a conditionally activebiologic protein, the method comprising: selecting a wild-type biologicprotein; evolving the DNA which encodes the wild-type biologic proteinusing one or more evolutionary techniques to create a mutant DNA;expressing the mutant DNA to obtain a mutant protein; subjecting themutant protein and the wild-type protein to an assay under a normalphysiological condition and the assay under an aberrant condition; andselecting the conditionally active biologic protein from those mutantproteins which exhibit both (a) a decrease in activity in the assay atthe normal physiological condition compared to the wild-type protein,and (b) an increase in activity in the assay under the aberrantcondition compared to the wild-type protein. In various aspects, thenormal physiological condition is selected from one or more oftemperature, pH, osmotic pressure, osmolality, oxidation and electrolyteconcentration. In a particular aspect, the normal physiologicalcondition is temperature; wherein the conditionally active biologicprotein is virtually inactive at the normal physiological temperature,but is active at an aberrant temperature less than the normalphysiological temperature. In other aspects, the conditionally activebiologic protein is reversibly or irreversibly inactivated at the wildtype normal physiological conditions. In one specific aspect, theprotein is reversibly inactivated at the wild type normal physiologicalconditions. Alternatively, conditionally active biologic proteins areselected from those proteins which exhibit changes in activity,reversibly or irreversibly, in two or more different physiologicalconditions.

In one embodiment, the wild-type biologic protein is an enzyme. Incertain aspects, the wild-type biologic protein is selected from thegroup consisting of tissue plasminogen activator, streptokinase,urokinase, renin, and hyaluronidase.

In another embodiment, the wild-type biologic protein is selected fromcalcitonin gene-related peptide (CGRP), substance P(SP), neuropeptide Y(NPY), vasoactive intestinal peptide (VIP), vasopressin, andangiostatin.

In another embodiment, the biologic protein is an antibody.

In another embodiment, the disclosure provides a method of preparing aconditionally active biological response modifier, the methodcomprising: selecting an inflammatory response mediator; identifying awild-type antibody to the mediator; evolving the wild-type antibody;screening differentially for mutants that exhibit decreased binding tothe mediator relative to the wild-type antibody at a first condition,and exhibit increased binding affinity to the mediator at a secondcondition to identify up-mutants; and recombining the heavy chains andthe light chains of the up-mutants to create recombined up-mutants; andscreening the recombined up-mutants for mutants that exhibit decreasedbinding to the mediator relative to the wild-type antibody at the firstcondition, and show increased binding affinity to the mediator at thesecond condition to identify the conditionally active biologicalresponse modifier. In one aspect, the inflammatory response mediator isselected from IL-6, IL-6 receptor, TNF-alpha, IL-23 and IL-12. Inanother aspect, the first and second conditions are selected fromconditions of pH, osmotic pressure, osmolality, oxidation andelectrolyte concentration.

In another embodiment, the disclosure provides a pharmaceuticalcomposition comprising a conditionally active biologic protein, and apharmaceutically acceptable carrier.

DETAILED DESCRIPTION

In order to facilitate understanding of the examples provided herein,certain frequently occurring methods and/or terms will be described.

As used herein in connection with a measured quantity, the term “about”refers to the normal variation in that measured quantity that would beexpected by the skilled artisan making the measurement and exercising alevel of care commensurate with the objective of the measurement and theprecision of the measuring equipment used. Unless otherwise indicated,“about” refers to a variation of +/−10% of the value provided.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, an array of spatially localized compounds (e.g.,a VLSIPS peptide array, polynucleotide array, and/or combinatorial smallmolecule array), biological macromolecule, a bacteriophage peptidedisplay library, a bacteriophage antibody (e.g., scFv) display library,a polysome peptide display library, or an extract made from biologicalmaterials such as bacteria, plants, fungi, or animal (particularmammalian) cells or tissues. Agents are evaluated for potential enzymeactivity by inclusion in screening assays described herein below. Agentsare evaluated for potential activity as conditionally active biologictherapeutic enzymes by inclusion in screening assays described hereinbelow.

An “ambiguous base requirement” in a restriction site refers to anucleotide base requirement that is not specified to the fullest extent,i.e. that is not a specific base (such as, in a non-limitingexemplification, a specific base selected from A, C, G, and T), butrather may be any one of at least two or more bases. Commonly acceptedabbreviations that are used in the art as well as herein to representambiguity in bases include the following: R=G or A; Y=C or T; M=A or C;K=G or T; S=G or C; W=A or T; H=A or C or T; B=G or T or C; V=G or C orA; D=G or A or T; N=A or C or G or T.

The term “amino acid” as used herein refers to any organic compound thatcontains an amino group (—NH2) and a carboxyl group (—COOH); preferablyeither as free groups or alternatively after condensation as part ofpeptide bonds. The “twenty naturally encoded polypeptide-formingalpha-amino acids” are understood in the art and refer to: alanine (alaor A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp orD), cysteine (cys or C), gluatamic acid (glu or E), glutamine (gin orQ), glycine (gly or G), histidine (his or H), isoleucine (ile or I),leucine (leu or L), lysine (lys or K}, methionine (met or M),phenylalanine (phe or F), proline (pro or P), serine (ser or S),threonine (thr or T), tryptophan (trp or W), tyrosine (tyr or Y), andvaline (val or V).

The term “amplification” means that the number of copies of apolynucleotide is increased.

A molecule that has a “chimeric property” is a molecule that is: 1) inpart homologous and in part heterologous to a first reference molecule;while 2} at the same time being in part homologous and in partheterologous to a second reference molecule; without 3} precluding thepossibility of being at the same time in part homologous and in partheterologous to still one or more additional reference molecules. In anon-limiting embodiment, a chimeric molecule may be prepared byassembling a reassortment of partial molecular sequences. In anon-limiting aspect, a chimeric polynucleotide molecule may be preparedby synthesizing the chimeric polynucleotide using plurality of moleculartemplates, such that the resultant, chimeric polynucleotide hasproperties of a plurality of templates.

The term “cognate” as used herein refers to a gene sequence that isevolutionarily and functionally related between species. For example,but not limitation, in the human genome the human CD4 gene is thecognate gene to the mouse 3d4 gene, since the sequences and structuresof these two genes indicate that they are highly homologous and bothgenes encode a protein which functions in signaling T cell activationthrough MHC class II-restricted antigen recognition.

A “comparison window,” as used herein, refers to a conceptual segment ofat least 20 contiguous nucleotide positions wherein a polynucleotidesequence may be compared to a reference sequence of at least 20contiguous nucleotides and wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Optimal alignment of sequences for aligning acomparison window may be conducted by the local homology algorithm ofSmith (Smith and Waterman, 1981, “Comparison of biosequences”, Adv ApplMath, 2:482-489; Smith and Waterman, 1981, “Overlapping genes andinformation theory”, J Theor Biol, 91:379-380; Smith and Waterman, J MolBioi, “Identification of common molecular subsequences”, 1981,147:195-197; Smith et al., 1981, ““Comparative biosequence metrics”, JMol Evol, 18:38-46), by the homology alignment algorithm of Needleman(Needleman and Wunsch, 1970, “A general method applicable to the searchfor similarities in the amino acid sequence of two proteins” J Mol Bioi,48(3):443-453), by the search of similarity method of Pearson (Pearsonand Lipman, 1988, “Improved tools for biological sequence comparison”,Proc Nat Acad Sci USA, 85:2444-2448), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package Release 7.0, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by inspection, and the best alignment(i.e., resulting in the highest percentage of homology over thecomparison window) generated by the various methods is selected.

The term “conditionally active biologic protein” refers to a variant, ormutant, of a wild-type protein which is more or less active than theparent wild-type protein under one or more normal physiologicalconditions. This conditionally active protein also exhibits activity inselected regions of the body and/or exhibits increased or decreasedactivity under aberrant, or permissive, physiological conditions. Normalphysiological conditions are those of temperature, pH, osmotic pressure,osmolality, oxidation and electrolyte concentration which would beconsidered within a normal range at the site of administration, or atthe tissue or organ at the site of action, to a subject. An aberrantcondition is that which deviates from the normally acceptable range forthat condition. In one aspect, the conditionally active biologic proteinis virtually inactive at wild-type conditions but is active at otherthan wild-type conditions at a level that is equal or better than atwild-type conditions. For example, in one aspect, an evolvedconditionally active biologic protein is virtually inactive at bodytemperature, but is active at lower temperatures. In another aspect, theconditionally active biologic protein is reversibly or irreversiblyinactivated at the wild type conditions. In a further aspect, thewild-type protein is a therapeutic protein. In another aspect, theconditionally active biologic protein is used as a drug, or therapeuticagent. In yet another aspect, the protein is more or less active inhighly oxygenated blood, such as, for example, after passage through thelung or in the lower pH environments found in the kidney.

“Conservative amino acid substitutions” refer to the interchangeabilityof residues having similar side chains. For example, a group of aminoacids having aliphatic side chains is glycine, alanine, valine, leucine,and isoleucine; a group of amino acids having aliphatic-hydroxyl sidechains is serine and threonine; a group of amino acids havingamide-containing side chains is asparagine and glutamine; a group ofamino acids having aromatic side chains is phenylalanine, tyrosine, andtryptophan; a group of amino acids having basic side chains is lysine,arginine, and histidine; and a group of amino acids havingsulfur-containing side chains is cysteine and methionine. Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyro sine, lysine-arginine,alanine-valine, and asparagine-glutamine.

The term “corresponds to” is used herein to mean that a polynucleotidesequence is homologous (i.e., is identical, not strictly evolutionarilyrelated) to all or a portion of a reference polynucleotide sequence, orthat a polypeptide sequence is identical to a reference polypeptidesequence. In contradistinction, the term “complementary to” is usedherein to mean that the complementary sequence is homologous to all or aportion of a reference polynucleotide sequence. For illustration, thenucleotide sequence “TATAC” corresponds to a reference “TATAC” and iscomplementary to a reference sequence “GTATA.”

The term “degrading effective” amount refers to the amount of enzymewhich is required to process at least 50% of the substrate, as comparedto substrate not contacted with the enzyme.

As used herein, the term “defined sequence framework” refers to a set ofdefined sequences that are selected on a non-random basis, generally onthe basis of experimental data or structural data; for example, adefined sequence framework may comprise a set of amino acid sequencesthat are predicted to form a .beta.-sheet structure or may comprise aleucine zipper heptad repeat motif, a zinc-finger domain, among othervariations. A “defined sequence kemal” is a set of sequences whichencompass a limited scope of variability. Whereas (1) a completelyrandom 10-rner sequence of the 20 conventional amino acids can be any of(20)¹⁰ sequences, and (2) a pseudorandom 10-rner sequence of the 20conventional amino acids can be any of (20)¹⁰ sequences but will exhibita bias for certain residues at certain positions and/or overall, (3) adefined sequence kernal is a subset of sequences if each residueposition was allowed to be any of the allowable 20 conventional aminoacids (and/or allowable unconventional amino/imino acids). A definedsequence kernal generally comprises variant and invariant residuepositions and/or comprises variant residue positions which can comprisea residue selected from a defined subset of amino acid residues, and thelike, either segmentally or over the entire length of the individualselected library member sequence. Defined sequence kernels can refer toeither amino acid sequences or polynucleotide sequences. Of illustrationand not limitation, the sequences (NNK)w and (NNM)w, wherein Nrepresents A, T, G, or C; K represents G or T; and M represents A or C,are defined sequence kernels.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 microgram of plasmid or DNA fragment is used withabout 2 units of enzyme in about 20 microliters of buffer solution. Forthe purpose of isolating DNA fragments for plasmid construction,typically 5 to 50 micrograms of DNA are digested with 20 to 250 units ofenzyme in a larger volume. Appropriate buffers and substrate amounts forparticular restriction enzymes are specified by the manufacturer.Incubation times of about 1 hour at 37 degrees C. are ordinarily used,but may vary in accordance with the supplier's instructions. Afterdigestion the reaction is electrophoresed directly on a gel to isolatethe desired fragment.

“Directional ligation” refers to a ligation in which a 5′ end and a 3′end of a polynucleotide are different enough to specify a preferredligation orientation. For example, an otherwise untreated and undigestedPCR product that has two blunt ends will typically not have a preferredligation orientation when ligated into a cloning vector digested toproduce blunt ends in its multiple cloning site; thus, directionalligation will typically not be displayed under these circumstances. Incontrast, directional ligation will typically be displayed when adigested PCR product having a 5′ EcoR !-treated end and a 3′ BamH I isligated into a cloning vector that has a multiple cloning site digestedwith EcoR I and BamH I .

The term “DNA shuffling” is used herein to indicate recombinationbetween substantially homologous but non-identical sequences, in someembodiments DNA shuffling may involve crossover via non-homologousrecombination, such as via cer/lox and/or flp/frt systems and the like.DNA shuffling can be random or non-random.

The term “drug” or “drug molecule” refers to a therapeutic agentincluding a substance having a beneficial effect on a human or animalbody when it is administered to the human or animal body. Preferably,the therapeutic agent includes a substance that can treat, cure orrelieve one or more symptoms, illnesses, or abnormal conditions in ahuman or animal body or enhance the wellness of a human or animal body.

An “effective amount” is an amount of a conditionally active biologicprotein or fragment which is effective to treat or prevent a conditionin a living organism to whom it is administered over some period oftime, e.g., provides a therapeutic effect during a desired dosinginterval.

As used herein, the term “electrolyte” is used to define a mineral inthe blood or other body fluids that carries a charge. For example, inone aspect, the normal physiological condition and aberrant conditioncan be conditions of electrolyte concentration”. In one aspect, theelectrolyte concentration to be tested is selected from one or more ofionized calcium, sodium, potassium, magnesium, chloride, bicarbonate,and phosphate concentration. For example, in one aspect, normal range ofserum calcium is 8.5 to 10.2 mg/dL. In this aspect, aberrant serumcalcium concentration may be selected from either above or below thenormal range. In another example, in one aspect, normal range of serumchloride is 96-106 milliequivalents per liter (mEq/L). In this aspect,aberrant serum chloride concentration may be selected from either aboveor below the normal range. In another example, in one aspect, a normalrange of serum magnesium is from 1.7-2.2 mg/dL. In this aspect, anaberrant serum magnesium concentration may be selected from either aboveor below the normal range. In another example, in one aspect, a normalrange of serum phosphorus is from 2.4 to 4.1 mg/dL. In this aspect,aberrant serum phosphorus concentration may be selected from eitherabove or below the normal range. In another example, in one aspect, anormal range of serum, or blood, sodium is from 135 to 145 mEq/L. Inthis aspect, aberrant serum, or blood, sodium concentration may beselected from either above or below the normal range. In anotherexample, in one aspect, a normal range of serum, or blood, potassium isfrom 3.7 to 5.2 mEq/L. In this aspect, aberrant serum, or blood,potassium concentration may be selected from either above or below thenormal range. In a further aspect, a normal range of serum bicarbonateis from 20 to 29 mEq/L. In this aspect, aberrant serum, or blood,bicarbonate concentration may be selected from either above or below thenormal range. In a different aspect, bicarbonate levels can be used toindicate normal levels of acidity (pH), in the blood. The term“electrolyte concentration” may also be used to define the condition ofa particular electrolyte in a tissue or body fluid other than blood orplasma. In this case, the normal physiological condition is consideredto be the clinically normal range for that tissue or fluid. In thisaspect, aberrant tissue or fluid electrolyte concentration may beselected from either above or below the normal range.

As used in this disclosure, the term “epitope” refers to an antigenicdeterminant on an antigen, such as an enzyme polypeptide, to which theparatope of an antibody, such as an enzyme-specific antibody, binds.Antigenic determinants usually consist of chemically active surfacegroupings of molecules, such as amino acids or sugar side chains, andcan have specific three-dimensional structural characteristics, as wellas specific charge characteristics. As used herein “epitope” refers tothat portion of an antigen or other macromolecule capable of forming abinding interaction that interacts with the variable region binding bodyof an antibody. Typically, such binding interaction is manifested as anintermolecular contact with one or more amino acid residues of a CDR.

As used herein, an “enzyme” is a protein with specific catalyticproperties. Factors such as, for example, substrate concentration, pH,temperature and presence or absence of inhibitors can affect the rate ofcatalysis. Typically, for a wild type enzyme, Q10 (the temperaturecoefficient) describes the increase in reaction rate with a 10 degreeCrise in temperature. For wild type enzymes, the Q10=2 to 3; in otherwords, the rate of reaction doubles or triples with every 10 degreeincrease in temperature. At high temperatures, proteins denature. At pHvalues slightly different from an enzymes optimum value, small changesoccur in the charges of the enzyme and perhaps the substrate molecule.The change in ionization can affect the binding of the substratemolecule. At extreme pH levels, the enzyme will produce denaturation,where the active site is distorted, and the substrate molecule will nolonger fit.

As used herein, the term “evolution”, or “evolving”, refers to using oneor more methods of mutagenesis to generate a novel polynucleotideencoding a novel polypeptide, which novel polypeptide is itself animproved biological molecule &/or contributes to the generation ofanother improved biological molecule. In a particular non-limitingaspect, the present disclosure relates to evolution of conditionallyactive biologic proteins from a parent wild type protein. In one aspect,for example, evolution relates to a method of performing bothnon-stochastic polynucleotide chimerization and non-stochasticsite-directed point mutagenesis disclosed in U.S. patent applicationpublication 2009/0130718, which is incorporated herein by reference.More particularly, the present disclosure provides methods for evolutionof conditionally active biologic enzymes which exhibit reduced activityat normal physiological conditions compared to a wild-type enzyme parentmolecule, but enhanced activity under one or more aberrant conditionscompared to the wild-type enzyme.

The terms “fragment”, “derivative” and “analog” when referring to areference polypeptide comprise a polypeptide which retains at least onebiological function or activity that is at least essentially same asthat of the reference polypeptide. Furthermore, the terms “fragment”,“derivative” or “analog” are exemplified by a “pro-form” molecule, suchas a low activity proprotein that can be modified by cleavage to producea mature enzyme with significantly higher activity.

A method is provided herein for producing from a template polypeptide aset of progeny polypeptides in which a “full range of single amino acidsubstitutions” is represented at each amino acid position. As usedherein, “full range of single amino acid substitutions” is in referenceto the 20 naturally encoded polypeptide-forming alpha-amino acids, asdescribed herein.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

“Genetic instability”, as used herein, refers to the natural tendencyofhighly repetitive sequences to be lost through a process of reductiveevents generally involving sequence simplification through the loss ofrepeated sequences. Deletions tend to involve the loss of one copy of arepeat and everything between the repeats.

The term “heterologous” means that one single-stranded nucleic acidsequence is unable to hybridize to another single-stranded nucleic acidsequence or its complement. Thus areas of heterology means that areas ofpolynucleotides or polynucleotides have areas or regions within theirsequence which are unable to hybridize to another nucleic acid orpolynucleotide. Such regions or areas are for example areas ofmutations.

The term “homologous” or “homeologous” means that one single-strandednucleic acid sequence may hybridize to a complementary single-strandednucleic acid sequence. The degree of hybridization may depend on anumber of factors including the amount of identity between the sequencesand the hybridization conditions such as temperature and saltconcentrations as discussed later. Preferably the region of identity isgreater than about 5 bp, more preferably the region of identity isgreater than 10 bp.

The benefits of this disclosure extend to “industrial applications” (orindustrial processes), which term is used to include applications incommercial industry proper (or simply industry) as well asnon-commercial industrial applications (e.g. biomedical research at anon-profit institution). Relevant applications include those in areas ofdiagnosis, medicine, agriculture, manufacturing, and academia.

The term “identical” or “identity” means that two nucleic acid sequenceshave the same sequence or a complementary sequence. Thus, “areas ofidentity” means that regions or areas of a polynucleotide or the overallpolynucleotide are identical or complementary to areas of anotherpolynucleotide.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide or enzymepresent in a living animal is not isolated, but the same polynucleotideor enzyme, separated from some or all of the coexisting materials in thenatural system, is isolated. Such polynucleotides could be part of avector and/or such polynucleotides or enzymes could be part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment.

The term “isolated nucleic acid” is used to define a nucleic acid, e.g.,a DNA or RNA molecule, that is not immediately contiguous with the 5′and 3′ flanking sequences with which it normally is immediatelycontiguous when present in the naturally occurring genome of theorganism from which it is derived. The term thus describes, for example,a nucleic acid that is incorporated into a vector, such as a plasmid orviral vector; a nucleic acid that is incorporated into the genome of aheterologous cell (or the genome of a homologous cell, but at a sitedifferent from that at which it naturally occurs); and a nucleic acidthat exists as a separate molecule, e.g., a DNA fragment produced by PCRamplification or restriction enzyme digestion, or an RNA moleculeproduced by in vitro transcription. The term also describes arecombinant nucleic acid that forms part of a hybrid gene encodingadditional polypeptide sequences that can be used, for example, in theproduction of a fusion protein.

As used herein “ligand” refers to a molecule, such as a random peptideor variable segment sequence, that is recognized by a particularreceptor. As one of skill in the art will recognize, a molecule (ormacromolecular complex) can be both a receptor and a ligand. In general,the binding partner having a smaller molecular weight is referred to asthe ligand and the binding partner having a greater molecular weight isreferred to as a receptor.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Sambrook et al., (1982).Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratory,Cold Spring Harbor, N.Y., p. 146; Sambrook et al., Molecular Cloning: alaboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989).Unless otherwise provided, ligation may be accomplished using knownbuffers and conditions with 10 units of T4 DNA ligase (“ligase”) per 0.5micrograms of approximately equimolar amounts of the DNA fragments to beligated.

As used herein, “linker” or “spacer” refers to a molecule or group ofmolecules that connects two molecules, such as a DNA binding protein anda random peptide, and serves to place the two molecules in a preferredconfiguration, e.g., so that the random peptide can bind to a receptorwith minimal steric hindrance from the DNA binding protein.

As used herein “microenvironment” means any portion or region of atissue or body that has constant or temporal, physical or chemicaldifferences from other regions of the tissue or regions of the body.

As used herein, a “molecular property to be evolved” includes referenceto molecules comprised of a polynucleotide sequence, molecules comprisedof a polypeptide sequence, and molecules comprised in part of apolynucleotide sequence and in part of a polypeptide sequence.Particularly relevant—but by no means limiting—examples of molecularproperties to be evolved include protein activities at specifiedconditions, such as related to temperature; salinity; osmotic pressure;pH; oxidation, and concentration of glycerol, DMSO, detergent, &/or anyother molecular species with which contact is made in a reactionenvironment. Additional particularly relevant—but by no meanslimiting—examples of molecular properties to be evolved includestabilities—e.g. the amount of a residual molecular property that ispresent after a specified exposure time to a specified environment, suchas may be encountered during storage.

The term “mutations” means changes in the sequence of a wild-typenucleic acid sequence or changes in the sequence of a peptide. Suchmutations may be point mutations such as transitions or transversions.The mutations may be deletions, insertions or duplications.

As used herein, the degenerate “N,N,G/T” nucleotide sequence represents32 possible triplets, where “N” can be A, C, G or T.

The term “naturally-occurring” as used herein as applied to the objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally occurring. Generally, the term naturally occurring refers toan object as present in a non-pathological (un-diseased) individual,such as would be typical for the species.

As used herein, “normal physiological conditions”, or “wild typeoperating conditions”, are those conditions of temperature, pH, osmoticpressure, osmolality, oxidation and electrolyte concentration whichwould be considered within a normal range at the site of administration,or the site of action, in a subject.

As used herein, a “nucleic acid molecule” is comprised of at least onebase or one base pair, depending on whether it is single-stranded ordouble-stranded, respectively. Furthermore, a nucleic acid molecule maybelong exclusively or chimerically to any group of nucleotide-containingmolecules, as exemplified by, but not limited to, the following groupsof nucleic acid molecules: RNA, DNA, genomic nucleic acids, non-genomicnucleic acids, naturally occurring and not naturally occurring nucleicacids, and synthetic nucleic acids. This includes, by way ofnon-limiting example, nucleic acids associated with any organelle, suchas the mitochondria, ribosomal RNA, and nucleic acid molecules comprisedchimerically of one or more components that are not naturally occurringalong with naturally occurring components.

Additionally, a “nucleic acid molecule” may contain in part one or morenon-nucleotide-based components as exemplified by, but not limited to,amino acids and sugars. Thus, by way of example, but not limitation, aribozyme that is in part nucleotide-based and in part protein-based isconsidered a “nucleic acid molecule”.

In addition, by way of example, but not limitation, a nucleic acidmolecule that is labeled with a detectable moiety, such as a radioactiveor alternatively a non-radioactive label, is likewise considered a“nucleic acid molecule”.

The terms “nucleic acid sequence coding for” or a “DNA coding sequenceof or a “nucleotide sequence encoding” a particular enzyme—as well asother synonymous terms—refer to a DNA sequence which is transcribed andtranslated into an enzyme when placed under the control of appropriateregulatory sequences. A “promotor sequence” is a DNA regulatory regioncapable of binding RNA polymerase in a cell and initiating transcriptionof a downstream (3′ direction) coding sequence. The promoter is part ofthe DNA sequence. This sequence region has a start codon at its 3′terminus. The promoter sequence does include the minimum number of baseswhere elements necessary to initiate transcription at levels detectableabove background. However, after the RNA polymerase binds the sequenceand transcription is initiated at the start codon (3′ terminus with apromoter), transcription proceeds downstream in the 3′ direction. Withinthe promotor sequence will be found a transcription initiation site(conveniently defined by mapping with nuclease S1) as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

The terms “nucleic acid encoding an enzyme (protein)” or “DNA encodingan enzyme (protein)” or “polynucleotide encoding an enzyme (protein)”and other synonymous terms encompasses a polynucleotide which includesonly coding sequence for the enzyme as well as a polynucleotide whichincludes additional coding and/or non-coding sequence.

In one preferred embodiment, a “specific nucleic acid molecule species”is defined by its chemical structure, as exemplified by, but not limitedto, its primary sequence. In another preferred embodiment, a specific“nucleic acid molecule species” is defined by a function of the nucleicacid species or by a function of a product derived from the nucleic acidspecies. Thus, by way of non-limiting example, a “specific nucleic acidmolecule species” may be defined by one or more activities or propertiesattributable to it, including activities or properties attributable toits expressed product.

The instant definition of“assembling a working nucleic acid sample intoa nucleic acid library” includes the process of incorporating a nucleicacid sample into a vector-based collection, such as by ligation into avector and transformation of a host. A description of relevant vectors,hosts, and other reagents as well as specific non-limiting examplesthereof are provided hereinafter. The instant definition of “assemblinga working nucleic acid sample into a nucleic acid library” also includesthe process of incorporating a nucleic acid sample into anon-vector-based collection, such as by ligation to adaptors. Preferablythe adaptors can anneal to PCR primers to facilitate amplification byPCR.

Accordingly, in a non-limiting embodiment, a “nucleic acid library” iscomprised of a vector-based collection of one or more nucleic acidmolecules. In another preferred embodiment a “nucleic acid library” iscomprised of a non-vector-based collection of nucleic acid molecules. Inyet another preferred embodiment a “nucleic acid library” is comprisedof a combined collection of nucleic acid molecules that is in partvector-based and in part non-vector-based. Preferably, the collection ofmolecules comprising a library is searchable and separable according toindividual nucleic acid molecule species.

The present disclosure provides a “nucleic acid construct” oralternatively a “nucleotide construct” or alternatively a “DNAconstruct”. The term “construct” is used herein to describe a molecule,such as a polynucleotide (e.g., an enzyme polynucleotide) which mayoptionally be chemically bonded to one or more additional molecularmoieties, such as a vector, or parts of a vector. In a specific—but byno means limiting—aspect, a nucleotide construct is exemplified by DNAexpression constructs suitable for the transformation of a host cell.

An “oligonucleotide” (or synonymously an “oligo”) refers to either asingle stranded polydeoxynucleotide or two complementarypolydeoxynucleotide strands which may be chemically synthesized. Suchsynthetic oligonucleotides may or may not have a 5′ phosphate. Thosethat do not will not ligate to another oligonucleotide without adding aphosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide will ligate to a fragment that has not beendephosphorylated. To achieve polymerase-based amplification (such aswith PCR), a “32-fold degenerate oligonucleotide that is comprised of,in series, at least a first homologous sequence, a degenerate N,N,G/Tsequence, and a second homologous sequence” is mentioned. As used inthis context, “homologous” is in reference to homology between the oligoand the parental polynucleotide that is subjected to thepolymerase-based amplification.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. A nucleic acid is“operably linked” when it is placed into a functional relationship withanother nucleic acid sequence. For instance, a promoter or enhancer isoperably linked to a coding sequence if it affects the transcription ofthe coding sequence. Operably linked means that the DNA sequences beinglinked are typically contiguous and, where necessary to join two proteincoding regions, contiguous and in reading frame.

A coding sequence is “operably linked to” another coding sequence whenRNA polymerase will transcribe the two coding sequences into a singlernRNA, which is then translated into a single polypeptide having aminoacids derived from both coding sequences. The coding sequences need notbe contiguous to one another so long as the expressed sequences areultimately processed to produce the desired protein.

As used herein the term “parental polynucleotide set” is a set comprisedof one or more distinct polynucleotide species. Usually this term isused in reference to a progeny polynucleotide set which is preferablyobtained by mutagenization of the parental set, in which case the terms“parental”, “starting” and “template” are used interchangeably.

The term “patient”, or “subject”, refers to an animal, for example amammal, such as a human, who is the object of treatment. The subject, orpatient, may be either male or female.

As used herein the term “physiological conditions” refers totemperature, pH, osmotic pressure, ionic strength, viscosity, and likebiochemical parameters which are compatible with a viable organism,and/or which typically exist intracellularly in a viable cultured yeastcell or mammalian cell. For example, the intracellular conditions in ayeast cell grown under typical laboratory culture conditions arephysiological conditions. Suitable in vitro reaction conditions for invitro transcription cocktails are generally physiological conditions. Ingeneral, in vitro physiological conditions comprise 50-200 mM NaCl orKCl, pH 6.5-8.5, 20-45 degrees C. and 0.001-10 mM divalent cation (e.g.,Mg++, Ca}; preferably about 150 mM NaCl or KCl, pH 7.2-7.6, 5 mMdivalent cation, and often include 0.01-1.0 percent nonspecific protein(e.g., BSA). A non-ionic detergent (Tween, NP-40, Triton X-100) canoften be present, usually at about 0.001 to 2%, typically 0.05-0.2%(v/v). Particular aqueous conditions may be selected by the practitioneraccording to conventional methods. For general guidance, the followingbuffered aqueous conditions maybe applicable: 10-250 mM NaCl, 5-50 mMTris HCl, pH 5-8, with optional addition of divalent cation(s) and/ormetal chelators and/or non-ionic detergents and/or membrane fractionsand/or anti-foam agents and/or scintillants. Normal physiologicalconditions refer to conditions of temperature, pH, osmotic pressure,osmolality, oxidation and electrolyte concentration in vivo in a patientor subject at the site of administration, or the site of action, whichwould be considered within the normal range in a patient.

Standard convention (5′ to 3′) is used herein to describe the sequenceof double standed polynucleotides.

The term “population” as used herein means a collection of componentssuch as polynucleotides, portions or polynucleotides or proteins. A“mixed population” means a collection of components which belong to thesame family of nucleic acids or proteins (i.e., are related) but whichdiffer in their sequence (i.e., are not identical) and hence in theirbiological activity.

A molecule having a “pro-form” refers to a molecule that undergoes anycombination of one or more covalent and noncovalent chemicalmodifications (e.g. glycosylation, proteolytic cleavage, dimerization oroligomerization, temperature-induced or pH-induced conformationalchange, association with a co-factor, etc.) en route to attain a moremature molecular form having a property difference (e.g. an increase inactivity) in comparison with the reference pro-form molecule. When twoor more chemical modifications (e.g. two proteolytic cleavages, or aproteolytic cleavage and a deglycosylation) can be distinguished enroute to the production of a mature molecule, the reference precursormolecule may be termed a “pre-pro-form” molecule.

As used herein, the term “pseudorandom” refers to a set of sequencesthat have limited variability, such that, for example, the degree ofresidue variability at another position, but any pseudorandom positionis allowed some degree of residue variation, however circumscribed.

“Quasi-repeated units”, as used herein, refers to the repeats to bere-assorted and are by definition not identical. Indeed the method isproposed not only for practically identical encoding units produced bymutagenesis of the identical starting sequence, but also thereassortment of similar or related sequences which may divergesignificantly in some regions. Nevertheless, if the sequences containsufficient homologies to be reasserted by this approach, they can bereferred to as “quasi-repeated” units.

As used herein “random peptide library” refers to a set ofpolynucleotide sequences that encodes a set of random peptides, and tothe set of random peptides encoded by those polynucleotide sequences, aswell as the fusion proteins that contain those random peptides.

As used herein, “random peptide sequence” refers to an amino acidsequence composed of two or more amino acid monomers and constructed bya stochastic or random process. A random peptide can include frameworkor scaffolding motifs, which may comprise invariant sequences.

As used herein, “receptor” refers to a molecule that has an affinity fora given ligand. Receptors can be naturally occurring or syntheticmolecules. Receptors can be employed in an unaltered state or asaggregates with other species. Receptors can be attached, covalently ornon-covalently, to a binding member, either directly or via a specificbinding substance. Examples of receptors include, but are not limitedto, antibodies, including monoclonal antibodies and antisera reactivewith specific antigenic determinants (such as on viruses, cells, orother materials), cell membrane receptors, complex carbohydrates andglycoproteins, enzymes, and hormone receptors.

“Recombinant” enzymes refer to enzymes produced by recombinant DNAtechniques, i.e., produced from cells transformed by an exogenous DNAconstruct encoding the desired enzyme. “Synthetic” enzymes are thoseprepared by chemical synthesis.

The term “related polynucleotides” means that regions or areas of thepolynucleotides are identical and regions or areas of thepolynucleotides are heterologous.

“Reductive reassortment”, as used herein, refers to the increase inmolecular diversity that is accrued through deletion (and/or insertion)events that are mediated by repeated sequences.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence,” “comparisonwindow,” “sequence identity,” “percentage of sequence identity,” and“substantial identity.”

A “reference sequence” is a defined sequence used as a basis for asequence comparison; a reference sequence may be a subset of a largersequence, for example, as a segment of a full-length cDNA or genesequence given in a sequence listing, or may comprise a complete cDNA orgene sequence. Generally, a reference sequence is at least 20nucleotides in length, frequently at least 25 nucleotides in length, andoften at least 50 nucleotides in length. Since two polynucleotides mayeach (1) comprise a sequence (i.e., a portion of the completepolynucleotide sequence) that is similar between the two polynucleotidesand (2) may further comprise a sequence that is divergent between thetwo polynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity.

“Repetitive fudex (RI)”, as used herein, is the average number of copiesof the quasi-repeated units contained in the cloning vector.

The term “restriction site” refers to a recognition sequence that isnecessary for the manifestation of the action of a restriction enzyme,and includes a site of catalytic cleavage. It is appreciated that a siteof cleavage may or may not be contained within a portion of arestriction site that comprises a low ambiguity sequence (i.e. asequence containing the principal determinant of the frequency ofoccurrence of the restriction site). Thus, in many cases, relevantrestriction sites contain only a low ambiguity sequence with an internalcleavage site (e.g. G/AATTC in the EcoR site) or an immediatelyadjacent cleavage site (e.g. /CCWGG in the EcoR II site). In othercases, relevant restriction enzymes [e.g. the Eco57 site orCTGAAG(16/14)] contain a low ambiguity sequence (e.g. the CTGAAGsequence in the Eco57 site) with an external cleavage site (e.g. in theN.sub.16 portion of the Eco57 I site). When an enzyme (e.g. arestriction enzyme) is said to “cleave” a polynucleotide, it isunderstood to mean that the restriction enzyme catalyzes or facilitatesa cleavage of a polynucleotide.

In a non-limiting aspect, a “selectable polynucleotide” is comprised ofa 5′ terminal region (or end region), an intermediate region (i.e. aninternal or central region), and a 3′ terminal region (or end region).As used in this aspect, a 5′ terminal region is a region that is locatedtowards a 5′ polynucleotide terminus (or a 5′ polynucleotide end); thusit is either partially or entirely in a 5′ half of a polynucleotide.Likewise, a 3′ terminal region is a region that is located towards a 3′polynucleotide terminus (or a 3′ polynucleotide end); thus it is eitherpartially or entirely in a 3′ half of a polynucleotide. As used in thisnon-limiting exemplification, there may be sequence overlap between anytwo regions or even among all three regions.

The term “sequence identity” means that two polynucleotide sequences areidentical (i.e., on a nucleotide-by-nucleotide basis) over the window ofcomparison. The term “percentage of sequence identity” is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, U, or I) occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison (i.e., thewindow size), and multiplying the result by 100 to yield the percentageof sequence identity. This “substantial identity”, as used herein,denotes a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence having at least 80 percent sequenceidentity, preferably at least 85 percent identity, often 90 to 95percent sequence identity, and most commonly at least 99 percentsequence identity as compared to a reference sequence of a comparisonwindow of at least 25-50 nucleotides, wherein the percentage of sequenceidentity is calculated by comparing the reference sequence to thepolynucleotide sequence which may include deletions or additions whichtotal 20 percent or less of the reference sequence over the window ofcomparison.

As known in the art “similarity” between two enzymes is determined bycomparing the amino acid sequence and its conserved amino acidsubstitutes of one enzyme to the sequence of a second enzyme. Similaritymay be determined by procedures which are well-known in the art, forexample, a BLAST program (Basic Local Alignment Search Tool at theNational Center for Biological Information).

The members of a pair of molecules (e.g., an antibody-antigen pair or anucleic acid pair) are said to “specifically bind” to each other if theybind to each other with greater affinity than to other, non-specificmolecules. For example, an antibody raised against an antigen to whichit binds more efficiently than to a non-specific protein can bedescribed as specifically binding to the antigen. (Similarly, a nucleicacid probe can be described as specifically binding to a nucleic acidtarget if it forms a specific duplex with the target by base pairinginteractions (see above).)

“Specific hybridization” is defined herein as the formation of hybridsbetween a first polynucleotide and a second polynucleotide (e.g., apolynucleotide having a distinct but substantially identical sequence tothe first polynucleotide), wherein substantially unrelatedpolynucleotide sequences do not form hybrids in the mixture.

The term “specific polynucleotide” means a polynucleotide having certainend points and having a certain nucleic acid sequence. Twopolynucleotides wherein one polynucleotide has the identical sequence asa portion of the second polynucleotide but different ends comprises twodifferent specific polynucleotides.

“Stringent hybridization conditions” means hybridization will occur onlyif there is at least 90% identity, preferably at least 95% identity andmost preferably at least 97% identity between the sequences. SeeSambrook et al., Molecular Cloning: a laboratory manual, 2nd Ed., ColdSpring Harbor Laboratory Press, 1989, which is hereby incorporated byreference in its entirety.

Also included in the disclosure are polypeptides having sequences thatare “substantially identical” to the sequence of an enzyme polypeptide.A “substantially identical” amino acid sequence is a sequence thatdiffers from a reference sequence only by conservative amino acidsubstitutions, for example, substitutions of one amino acid for anotherof the same class (e.g., substitution of one hydrophobic amino acid,such as isoleucine, valine, leucine, or methionine, for another, orsubstitution of one polar amino acid for another, such as substitutionof arginine for lysine, glutamic acid for aspartic acid, or glutaminefor asparagine).

Additionally a “substantially identical” amino acid sequence is asequence that differs from a reference sequence or by one or morenon-conservative substitutions, deletions, or insertions, particularlywhen such a substitution occurs at a site that is not the active site ofthe molecule, and provided that the polypeptide essentially retains itsbehavioural properties. For example, one or more amino acids can bedeleted from an enzyme polypeptide, resulting in modification of thestructure of the polypeptide, without significantly altering itsbiological activity. For example, amino- or carboxyl-terminal aminoacids that are not required for enzyme biological activity can beremoved. Such modifications can result in the development of smalleractive enzyme polypeptides.

The present disclosure provides a “substantially pure enzyme”. The term“substantially pure enzyme” is used herein to describe a molecule, suchas a polypeptide (e.g., an enzyme polypeptide, or a fragment thereof)that is substantially free of other proteins, lipids, carbohydrates,nucleic acids, and other biological materials with which it is naturallyassociated. For example, a substantially pure molecule, such as apolypeptide, can be at least 60%, by dry weight, the molecule ofinterest. The purity of the polypeptides can be determined usingstandard methods including, e.g., polyacrylamide gel electrophoresis(e.g., SDS-PAGE), column chromatography (e.g., high performance liquidchromatography (HPLC)), and amino-terminal amino acid sequence analysis.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual macromolecular species in the composition),and preferably substantially purified fraction is a composition whereinthe object species comprises at least about 50 percent (on a molarbasis) of all macromolecular species present. Generally, a substantiallypure composition will comprise more than about 80 to 90 percent of allmacromolecular species present in the composition. Most preferably, theobject species is purified to essential homogeneity (contaminant speciescannot be detected in the composition by conventional detection methods)wherein the composition consists essentially of a single macromolecularspecies. Solvent species, small molecules (<500 Daltons), and elementalion species are not considered macromolecular species.

The term “treating” includes: (1) preventing or delaying the appearanceof clinical symptoms of the state, disorder or condition developing inan animal that may be afflicted with or predisposed to the state,disorder or condition but does not yet experience or display clinical orsubclinical symptoms of the state, disorder or condition; (2) inhibitingthe state, disorder or condition (i.e., arresting, reducing or delayingthe development of the disease, or a relapse thereof in case ofmaintenance treatment, of at least one clinical or subclinical symptomthereof); and/or (3) relieving the condition (i.e., causing regressionof the state, disorder or condition or at least one of its clinical orsubclinical symptoms). The benefit to a patient to be treated is eitherstatistically significant or at least perceptible to the patient or tothe physician.

As used herein, the term “variable segment” refers to a portion of anascent peptide which comprises a random, pseudorandom, or defined kemalsequence. A “variable segment” refers to a portion of a nascent peptidewhich comprises a random pseudorandom, or defined kemal sequence. Avariable segment can comprise both variant and invariant residuepositions, and the degree of residue variation at a variant residueposition may be limited: both options are selected at the discretion ofthe practitioner. Typically, variable segments are about 5 to 20 aminoacid residues in length (e.g., 8 to 10), although variable segments maybe longer and may comprise antibody portions or receptor proteins, suchas an antibody fragment, a nucleic acid binding protein, a receptorprotein, and the like.

The term “variant” refers to polynucleotides or polypeptides of thedisclosure modified at one or more base pairs, codons, introns, exons,or amino acid residues (respectively) of a wild-type protein parentmolecule. Variants can be produced by any number of means includingmethods such as, for example, error-prone PCR, shuffling,oligonucleotide-directed mutagenesis, assembly PCR, sexual PCRmutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly, saturation mutagenesis and any combinationthereof. Techniques for producing variant proteins having reducedactivity compared to the wild-type protein at a normal physiologicalcondition of e.g., one or more conditions of temperature, pH, osmoticpressure, osmolality, oxidation and electrolyte concentration; andenhanced activity at an aberrant condition, are disclosed herein.Variants may additionally be selected for the properties of enhancedchemical resistance, and proteolytic resistance, compared to thewild-type protein.

As used herein, the term “wild-type” means that the polynucleotide doesnot comprise any mutations. A “wild type protein”, “wild-type protein”,“wild-type biologic protein”, or “wild type biologic protein”, refers toa protein which can be isolated from nature that will be active at alevel of activity found in nature and will comprise the amino acidsequence found in nature. The terms “parent molecule” and “targetprotein” also refer to the wild-type protein.

The term “working”, as in “working sample”, for example, is simply asample with which one is working. Likewise, a “working molecule”, forexample is a molecule with which one is working.

The present disclosure is directed to methods of engineering or evolvingproteins to generate new molecules that are reversibly or irreversiblyinactivated at the wild type condition, but active at non-normalconditions at the same or equivalent level as the wild-type condition.These new proteins are referred to as “Mirac” proteins herein. Miracproteins are particularly valuable for development of novel therapeuticsthat are active for short or limited periods of time within the host.This is particularly valuable where extended operation of the protein atthe given dose would be harmful to the host, but where limited activityis required to perform the desired therapy. Examples of beneficialapplications include topical or systemic treatments at high dose, aswell as localized treatments in high concentration. Inactivation underthe physiological condition can be determined by a combination of thedosing and the rate of inactivation of the protein. This condition basedinactivation is especially important for enzyme therapeutics wherecatalytic activity cause substantial negative effects in a relativelyshort period of time.

The present disclosure is also directed to methods of engineering orevolving proteins to generate new molecules that are different from wildtype molecules in that they are reversibly or irreversibly activated orinactivated over time, or activated or inactivated only when they are incertain microenvironments in the body, including in specific organs inthe body (such as the bladder or kidney).

Target Wild-Type Proteins

Any therapeutic protein can serve as a target protein, or wild-typeprotein, for production of a conditionally active biologic protein. Inone aspect, the target protein is a wild-type enzyme. Currently usedtherapeutic enzymes include urokinase and streptokinase, used in thetreatment of blood clots; and hyaluronidase, used as an adjuvant toimprove the absorption and dispersion of other drugs. In one aspect, thewild-type protein selected for generation of a conditionally activebiologic protein can be a currently used therapeutic enzyme, in order toavoid or minimize deleterious side effects associated with the wild-typeprotein or enzyme. Alternatively, an enzyme not in current usage as atherapeutic can be selected for generation of a conditionally activebiologic protein. Certain non-limiting examples will be discussed infurther detail below.

Therapeutic proteins are those which can be used in medicine eitheralone or in conjunction with other therapies to treat various diseasesor medical conditions. The conditionally active biologic proteins of thedisclosure could be appropriate for use in one or more indicationsincluding the treatment of circulatory disorders, arthritis, multiplesclerosis, autoimmune disorders, cancer, dermatologic conditions and usein various diagnostic formats. Depending on the protein and indication,the conditionally active biologic enzyme protein could be administeredin parenteral, topical or oral formulations as discussed below.

Circulatory Disorders-Thrombosis and Thrombolytic Therapy.

A thrombus (blood clot) is defined as a solid mass derived from bloodconstituents that forms in the circulatory system. The thrombus isformed by a series of events involving blood coagulation factors,platelets, red blood cells, and interactions with the vessel wall. Aplatelet is an intravascular aggregation of platelets, fibrin andentrapped blood cells which can cause vascular obstruction. Byobstructing or blocking blood flow, the thrombus deprives downstreamtissue of oxygen supply. Fragments (emboli) of the thrombus may breakaway and obstruct smaller vessels. Arterial thrombus formation isprecipitated by any of a variety of factors including an underlyingstenosis-atherosclerosis, a low flow state-cardiac function,hypercoagubility as in cancer or a coagulation factor deficiency, or aforeign body such as a stentor catheter. A thrombus leading to arterialischemia can result in limb or tissue injury, acute myocardialinfarction (AMI), stroke, amputation, or bowel infarction. Major causesof morbidity and mortality are the formation of arterial thrombi(coronary arterial thrombi and cerebral arterial thrombi) and pulmonarythrombi. Venous thrombus formation can occur due to endothelial injurysuch as trauma, stasis due to e.g. immobility, or hypercoagulability,but atherosclerosos is not a factor. Treatment strategies includemechanical thrombectomy, pharmacomechanical thrombectomy andthrombolysis. Thrombotic therapy is used to minimize formation and aidin removal of thrombi.

Thrombotic therapy includes the use of antiplatelet agents which inhibitplatelet activation, anticoagulant therapies, and/or thrombolytictherapy to degrade blood clots. Examples of antiplatelets includeaspirin, dipyridamole, and ticlopidine. Examples of anticoagulantsinclude heparin, warfarin, hirudin, and activated human protein C.Examples of thrombolytics include tissue plasminogen activator (tPA)/tPAvariants, urokinase and streptokinase. The thrombolytics display acatalytic mode of action.

Thrombolytic therapy in acute myocardial infarction is well established.Use of thrombolytic agents has become standard emergency treatment.Although effective, these products achieve complete reperfusion in onlyabout 50% of patients and side effects include risk of hemorrhage (inparticular intracranial bleeding) as well as hypertension. Thedegradation of blood clots from a damaged or diseased vessel is termed“fibrinolysis” or the “fibrinolytic process”. Fibrinolysis is aproteolytic process, by a plasminogen activator which activates theprotein plasminogen, thereby forming plasmin. Plasmin proteolyticallydegrades the fibrin strands of the blood clot to dissolve the clot.Fibrin specific plasminogen activators include tissue plasminogenactivators or variants. Non-specific plasminogen activators can includestreptokinase and urokinase.

Certain commonly used thrombolytic therapies utilize one of severalavailable tissue plasminogen activator (tPA) variants. For example, tPAbased product variants which have been previously approved for use areAlteplase (rt-PA), Reteplase (r-PA) and Tenecteplase (TNK). Approveduses for tPA variants include, for example, acute myocardial infarctionfor the improvement of ventricular function following AMI, the reductionof incidence of congestive heart failure, and reduction of mortalityassociated with AMI, management of ischemic stroke in adults forimproving neurological recovery and reducing incidence of disability,management of acute massive pulmonary embolism in adults for the lysisof acute pulmonary emboli, and for the lysis of pulmonary emboliaccompanied by unstable hemodynamics.

Another commonly used thrombolytic therapy utilizes urokinase. Urokinaseis a standard lytic agent used in the management of peripheral vasculardisease.

Streptokinase is a protein secreted by several species of streptococcithat can bind and activate human plasminogen. Complexes of streptokinasewith human plasminogen can hydrolytically activate other unboundplasminogen by activating through bond cleavage to produce plasmin. Theusual activation of plasminogen is through the proteolysis of theArg561-Val562 bond. The amino group of Val562 then forms a salt-bridgewith Asp740, which causes a conformational change to produce the activeprotease plasmin. Plasmin is produced in the blood to break down fibrin,the major constituent of blood clots.

Streptokinase is used as an effective clot-dissolving medication in somecases of myocardial infarction (heart attack), pulmonary embolism (lungblood clots), and deep venous thrombosis (leg blood clots).Streptokinase belongs to a group of medications called fibrinolytics.Streptokinase is given as soon as possible after the onset of a heartattack to dissolve clots in the arteries of the heart wall and reducedamage to the heart muscle. Streptokinase is a bacterial product, so thebody has the ability to build up immunity against the protein.Therefore, it is recommended that this product should not be given againafter four days from the first administration, as it may not be aseffective and cause an allergic reaction. For this reason it is usuallygiven only after a first heart attack, and further thrombotic events aretypically treated with tissue plasminogen activator {TPA). Streptokinaseis also sometimes used to prevent post-operative adhesions.

Side effects of streptokinase include bleeding (major and minor),hypotension, and respiratory depression as well as possible allergicreaction. In addition, anticoagulants, agents that alter plateletfunction (e.g. aspirin, other NSAIDs, dipyridamole) may increase risk ofbleeding.

Administration of the thrombolytics is generally by infusion or by bolusintravenous dose; or by a mechanical infusion system. Adverse effectscan include serious intracranial, gastrointestinal, retroperitoneal, orpericardia! bleeding. If bleeding occurs the administration must bediscontinued immediately.

fu certain embodiments of the disclosure, tPA, streptokinase orurokinase is selected as the target, or wild-type protein.

In one embodiment, the methods of the disclosure are used to select fora conditionally active recombinant or synthetic streptokinase variantwith high activity at aberrant temperature conditions below normalphysiological conditions; and substantial deactivation or inactivationat normal physiological conditions (e.g. 37 degrees C.). In one aspect,the aberrant temperature condition is room temperature, e.g. 20-25degrees C. In another aspect, the disclosure provides a method oftreating a stroke or heart attack, the method comprising administering ahigh dose of the conditionally active streptokinase variant to stroke orheart attack victims in order to clear clots, yet allow for rapidinactivation of the streptokinase variant to avoid excessive bleeding.

Circulatory Disorders-Renin/Angiotensin

The renin-angiotensin system is a hormone system that regulates bloodpressure and water (fluid) balance. The kidneys secrete renin when theblood volume is low. Renin is an enzyme which hydrolyzes angiotensinogensecreted from the liver into the peptide angiotensin I. Angiotensin I isfurther cleaved in the lungs by endothelial-bound angiotensin convertingenzyme (ACE) into angiotensin IT, the most vasoactive peptide.Angiotensin II causes the blood vessels to constrict, resulting inincreased blood pressure. However, angiotensin II also stimulates thesecretion of the hormone aldosterone from the adrenal cortex.Aldosterone causes the tubules of the kidneys to increase the resorptionof sodium and water. This increases the volume of fluid in the body,which also increases blood pressure. An over-active renin-angiotensinsystem leads to vasoconstriction and retention of sodium and water.These effects lead to hypertension. There are many drugs which interruptdifferent steps in this system to lower blood pressure. These drugs areone of the main ways to control high blood pressure (hypertension),heart failure, kidney failure, and harmful effects of diabetes.

Hypovolemic shock is an emergency condition in which severe blood and/orfluid loss makes the heart unable to adequately perfuse the body's cellswith oxygenated blood. Blood loss can be from trauma, injuries andinternal bleeding. The amount of circulating blood may drop due toexcessive fluid loss from burns, diarrhea, excessive perspiration orvomiting. Symptoms of hypovolemic shock include anxiety, cool clammyskin, confusion, rapid breathing, or unconsciousness. Examination showssigns of shock including low blood pressure, low body temperature, andrapid pulse, which may be weak or thready. Treatment includesintravenous fluids; blood or blood products; treatment for shock; andmedication such as dopamine, dobutamine, epinephrine and norepinephrineto increase blood pressure and cardiac output.

In one embodiment, the disclosure provides a method of selecting for aconditionally active recombinant renin variant to be reversiblydeactivated at normal physiological temperature, but reactivated at theaberrant lower temperatures in a patient with hypovolemic shock. Theconditionally active protein can be used to treat hypovolemic shock tohelp increase the volume of fluid in the body, and increase bloodpressure.

Circulatory Disorders-Reynaud's Phenomenon

Reynaud's phenomenon (RP) is a vasospastic disorder causingdiscoloration of the fingers, toes and occasionally other extremities.Emotional stress and cold are classic triggers of the phenomenon. Whenexposed to cold temperatures, the extremities lose heat. The bloodsupply to fingers and toes is normally slowed to preserve the body'score temperature. Blood flow is reduced by the narrowing of smallarteries under the skin of the extremities. Stress causes similarreaction to cold in the body. In Reynaud's, the normal response isexaggerated. The condition can cause pain, discoloration, and sensationsof cold and numbness. The phenomenon is the result of vasospasms thatdecrease the blood supply to the respective regions. In Reynaud'sdisease (Primary Raynaud's phenomenon), the disease is idiopathic. InRaynaud's syndrome (Secondary Reynaud's), the phenomenon is caused bysome other instigating factor. Measurement of hand-temperature gradientsis one tool to distinguish between the primary and secondary forms. Theprimary form can progress to the secondary form, and in extreme cases,the secondary form can progress to necrosis or gangrene of thefingertips.

Raynaud's phenomenon is an exaggeration of responses to cold oremotional stress. Primary RP is essentially mediated by microvascularvasospasm. Hyperactivation of the sympathetic system causes extremevasoconstriction of the peripheral blood vessels, leading to hypoxia.Chronic, recurrent cases can result in atrophy of the skin, subcutaneoustissue, and muscle. It can also rarely result in ulceration and ischemicgangrene.

Traditional treatment options for Reynaud's phenomenon includeprescription medication that dilates blood vessels and promotescirculation. These include calcium channel blockers, such as nifedipineor diltiazem; alpha blockers, which counteract the actions ofnorepinephrine, a hormone that constricts blood vessels, such asprazosin or doxazosin; and vasodilators, to relax blood vessels, such asnitroglycerin cream, or the angiotensin II inhibitor losartan,sildenafil, or prostaglandins. Fluoxetine, a selective serotoninreuptake inhibitor and other antidepressant medications may reduce thefrequency and severity of episodes due to psychological stressors. Thesedrugs may cause side effects such as headache, flushing and ankle edema.A drug may also lose effectiveness over time.

The regulation of cutaneous vasoconstriction and vasodilation involvesaltered sympathetic nerve activity and a number of neuronal regulators,including adrenergic and non-adrenergic, as well as REDOX signaling andother signaling such as the RhoA/ROCK pathway. Vasoconstriction ofvascular smooth muscle cells (vSMC) in the skin is thought to beactivated by norepinephrine mediated by alpha1 and alpha2adrenoreceptors. Alpha2C-AR.s translocate from the trans Golgi to thecell surface of the vSMC where they respond to stimulation and signalingof these responses involves the RhoA/Rhokinase (ROCK) signaling pathway.Cold stimulation in cutaneous arteries results in the immediategeneration of reactive oxygen species (ROS) in the vSMC mitochondria.ROS are involved in the REDOX signaling through the RhoA/ROCK pathway.RhoA is a GTP-binding protein whose role is the regulation ofactin-myosin dependent processes such as migration and cell contractionin vSMC. Non-adrenergic neuropeptides with known function in vasculaturewith possible involvement in RP include calcitonin gene-related peptide(CGRP), Substance P(SP), Neuropeptide Y (NPY), and vasoactive intestinalpeptide(VIP). Fonseca et al., 2009, “Neuronal regulators and vasculardysfunction in Raynaud's phenomenon and systemic sclerosis”, Curr.Vascul. Pharmacol. 7:34-39.

New therapies for RP include alpha-2c adrenergic receptor blockers,protein tyrosine kinase inhibitors, Rho-kinase inhibitors and calcitoningene related peptide.

Calcitonin gene related peptide (CGRP) is a member of the calcitoninfamily of peptides and exists in two forms; alpha-CGRP and beta-CGRP.Alpha-CGRP is a 37-amino acid peptide formed from alternative splicingof the calcitonin/CGRP gene. CGRP is one of the most abundant peptidesproduced in peripheral and central neurons. It is a potent peptidevasodilator and can function in the transmission of pain. Migraine is acommon neurological disorder that is associated with an increase in CGRPlevels. CGRP dilates intracranial blood vessels and transmits vascularnociception. CGRP receptor antagonists have been tested as treatmentsfor migraines. Arulmani et al., 2004, “Calcitonin gene-related peptideand it role in migraine pathophysiology”, Eur. J. Pharmacol. 500(1-3):315-330. At least three receptor subtypes have been identified and CGRPacts through G protein-coupled receptors whose presence and changes infunction modulate the peptide's effect in various tissues. CGRP's signaltransduction through the receptors is dependent on two accessoryproteins: receptor activity modifying protein 1 (RAMP1) and receptorcomponent protein (RCP). Ghatta 2004, Calcitonin gene-related peptide:understanding its role. Indian J. Pharmacol. 36(5): 277-283. One studyof the effects of intravenous infusion of three vasodilators:endothelium-dependent vasodilator adenosine triphosphate (ATP),endothelium-independent vasodilator prostacyclin (epoprostenol; PGI2),and CGRP, to patients with Reynaud's phenomenon, and a similar number ofage and sex matched controls, using laser Doppler flowmetry (LDF) showedCGRP induced flushing of the face and hands by a rise in skin blood flowin the Reynaud's patients, whereas in controls CGRP caused flushing onlyin the face. PGI2 caused similar rises in blood flow in hands and faceof both groups. ATP did not cause any significant changes in blood flowin hands or face of the patients, but increased blood flow to the faceof controls. Shawket et al., 1989, “Selective suprasensitivity tocalcitonin-gene-related peptide in the hands in Reynaud's phenomenon”.The Lancet, 334(8676):1354-1357. In one aspect, the wild-type proteintarget molecule is CGRP.

In one embodiment, the disclosure provides methods of selecting forconditionally active recombinant protein variants of proteins associatedwith Reynaud's syndrome to be reversibly deactivated at normalphysiological temperature, but reactivated at the aberrant lowertemperatures in digits. The conditionally active proteins can be used totreat Reynaud's phenomenon, to prevent or reduce loss of digit functiondue to low circulation.

Circulatory Disorders-Vasopressin

Arginine vasopressin (AVP, vasopressin, antidiuretic hormone (ADH)) is apeptide hormone found in most mammals that controls reabsorption ofmolecules in the tubules of the kidney by affecting tissue permeability.One of the most important roles of vasopressin is to regulate waterretention in the body. In high concentrations it raises blood pressureby introducing moderate vasoconstriction. Vasopressin has three effectswhich result in increased urine osmolality (increased concentration) anddecreased water excretion. First, vasopressin causes an increase in thepermeability of water of the collecting duct cells in the kidneyallowing water resorption and excretion of a smaller volume ofconcentrated urine (antidiuresis). This occurs through insertion ofaquaporin-2 water channels into the apical membrane of the collectingduct cells. Secondly, vasopressin causes an increase in the permeabilityof the inner medullary portion of the collecting duct to urea, allowingincreased reabsorption urea into the medullary interstitium. Thirdly,vasopressin causes stimulation of sodium and chloride reabsorption inthe thick ascending limb of the loop of Henle by increasing the activityof the Na+-K+-2Cr-cotransporter. NaCl reabsorption drives the process ofcountercurrent multiplication, which furnishes the osmotic gradient foraquaporin mediated water reabsorption in the medullary collecting ducts.

The hypertonic interstitial fluid surrounding the collecting ducts ofthe kidney provides a high osmotic pressure for the removal of water.Transmembrane channels made of proteins called aquaporins are insertedin the plasma membrane greatly increasing its permeability to water.When open, an aquaporin channel allows 3 billion molecules of water topass through each second. Insertion of aquaporin-2 channels requiressignaling by vasopressin. Vasopressin binds to receptors (called V2receptors) on the basolateral surface of the cells of the collectingducts. Binding of the hormone triggers a rising level of cAMP within thecell. This “second messenger” initiates a chain of events culminating inthe insertion of aquaporin-2 channels in the apical surface of thecollecting duct cells. The aquaporins allow water to move out of thenephron, increasing the amount of water re-absorbed from the formingurine back into the bloodstream.

The main stimulus for the release of vasopressin from the pituitarygland is increased osmolality of the blood plasma. Anything thatdehydrates the body, such as perspiring heavily increases the osmoticpressure of the blood and turns on the vasopressin to V2 receptor toaquaporin-2 pathway. As a result, as little as 0.5 liters/day of urinemay remain of the original 180 liters/day of nephric filtrate. Theconcentration of salts in urine can be as high as four times that of theblood. If the blood should become too dilute, as would occur fromdrinking a large amount of water, vasopressin secretion is inhibited andthe aquaporin-2 channels are taken back into the cell by endocytosis.The result is that a large volume of watery urine is formed with a saltconcentration as little as one-fourth of that of the blood.

Decreased vasopressin release or decreased renal sensitivity to AVPleads to diabetes insipidus, a condition featuring hypematremia(increased blood sodium concentration), polyuria (excess urineproduction), and polydipsia (thirst).

High levels of AVP secretion (syndrome of inappropriate antidiuretichormone, SIADH) and resultant hyponatremia (low blood sodium levels)occurs in brain diseases and conditions of the lungs (Small cell lungcarcinoma). In the perioperative period, the effects of surgical stressand some commonly used medications (e.g., opiates, syntocinon,anti-emetics) lead to a similar state of excess vasopressin secretion.This may cause mild hyponatremia for several days.

Vasopressin agonists are used therapeutically in various conditions, andits long-acting synthetic analogue desmopressin is used in conditionsfeaturing low vasopressin secretion, as well as for control of bleeding(in some forms of von Willebrand disease) and in extreme cases ofbedwetting by children. Terlipressin and related analogues are used asvasoconstrictors in certain conditions. Vasopressin infusion has beenused as a second line of management in septic shock patients notresponding to high dose of inotropes (e.g., dopamine or norepinephrine).A vasopressin receptor antagonist is an agent that interferes withaction at the vasopressin receptors. They can be used in the treatmentof hyponatremia.

In one embodiment, the disclosure provides methods to select forconditionally active biologic recombinant or synthetic protein variantsof proteins involved in the vasopressin response to be reversiblydeactivated at normal physiological osmotic pressure, but reactivated ataberrant osmotic pressure in the blood. In another embodiment, variantsof proteins involved in the vasopressin response are activated underhyponatremic conditions, but inactivated at normal serum sodiumconcentrations. In one aspect, hyponatremic conditions are those whereserum sodium <135 mEq!L.

Cancer-Angiostatin

Angiostatin is a naturally occurring protein in several animal species.It acts as an endogenous angiogenesis inhibitor (i.e., it blocks thegrowth of new blood vessels). Angiostatin is able to suppress tumor cellgrowth and metastasis through inhibition of endothelial cellproliferation and migration. Angiostatin is a 38 kD fragment of plasmin(which is itself a fragment of plasminogen). Angiostatin comprises thekringles 1 to 3 of plasminogen. Angiostatin is produced, for example, byautolytic cleavage of plasminogen, involving extr cellular disulfidebond reduction by phosphoglycerate kinase. Angiostatin can also becleaved from plasminogen by different matrix metalloproteinases (MMPs)including MMP2, MMP12 and MMP9, and serine proteases (neutrophilelastase, prostate-specific antigen (PSA)). In vivo angiostatin inhibitstumor growth and keeps experimental metastasis in a dormant state.Angiostatin is elevated in animals with primary tumors and otherinflammatory and degenerative diseases.

Angiostatin is known to bind many proteins including angiomotin andendothelial cell surface ATO synthase, but also integrins, annexin IT,C-met receptor, NG2-proteoglycans, tissue-plasminogen activator,chondroitin sulfate glycoproteins, and CD26. One study shows that IL-12,a TH1 cytokine with potent antiangiogenic activity, is a mediator ofangiostatin's activity. Albin”., J. Translational Medicine. Jan. 4,2009, 7:5. Angiostatin binds and inhibits ATP synthase on theendothelial cell surface. ATP synthase also occurs on the surface of avariety of cancer cells. Tumor cell surface ATP synthase was found to bemore active at low extracellular pH; a hallmark of tumormicroenvironment. Angiostatin was found to affect tumor cell surface ATPsynthase activity at acidic extracellular pH (pHe). At low extracellularpH, angiostatin was directly anti-tumorigenic. At low pH, angiostatinand anti-beta-subunit antibody induce intracellular acidification ofA549 cancer cells, as well as a direct toxicity that is absent in tumorcells with low levels of extracellular ATP synthase. It was hypothesizedthat the mechanism of tumor cytotoxicity is dependent on intracellularpH deregulation due to inhibition of cell surface ATP synthase. Chi andPizzo, “Angiostatin is directly cytotoxic to tumor cells at lowextracellular pH: a mechanism dependent on cell surface-associated ATPsynthase”, Cancer Res., 2006, 66(2): 875-82.

In one embodiment, the disclosure provides a method for identificationof conditionally active angiostatin variant which is less active thanwild-type angiostatin at normal physiological blood pH, but exhibitsenhanced activity at low pH. Low pH is defined as being less than normalphysiological pH. In one aspect, low pH is less than about pH 7.2. In aparticular aspect, low pH is about pH 6.7.

In one aspect, the conditionally active angiostatin variant can beformulated and utilized as an anticancer agent.

Enhancement of Tissue Permeability-Hyaluronidase

Hyaluronidases are a family of enzymes that degrade hyaluronic acid. Bycatalyzing the degradation of hyaluronic acid, a major constituent ofthe interstitial barrier, hyaluronidase lowers the viscosity ofhyaluronic acid, thereby increasing tissue permeability. It is used inmedicine in conjunction with drugs to speed their dispersion anddelivery. The most common application is in ophthalmic surgery, used incombination with local anesthetics. Animal derived hyaluronidase includeHydase™ (PrimaPharm Inc.; Alcorn Inc.), Vitrase (ISTA Pharmaceuticals)and Amphadase (Amphastar Pharmaceuticals). Human RecombinantHyaluronidase is currently approved as an adjuvant to increaseabsorption of other drugs; hypodermocyclis (subcutaneous infusion offluids); adjunct in subcutaneous urography to improve resorption ofradioopaque agents. (Hylenex; Halozyme Therapeutics, Inc.; BaxterHealthcare Corp.) In one embodiment, hyaluronidase can serve as awild-type protein (parent molecule) for preparation of a conditionallyactive biologic protein. Hyaluronidases may play a role in cancermetastasis and perhaps angiogenesis; therefore overexposure to theseenzymes could be deleterious. In one aspect, a conditionally activebiologic hyaluronidase protein would become irreversibly or reversiblyinactivated at normal physiological temperature, but would be active ata level equal to or exceeding that of the wild-type hyaluronidase atcertain temperature ranges below that of normal physiologicaltemperature.

Autoimmune Diseases-Conditionally Active Biological Response Modifiers

Rheumatoid arthritis is an autoimmune disease characterized by aberrantimmune mechanisms that lead to joint inflammation and swelling withprogressive destruction of the joints. RA can also affect the sconnective tissue and organs in the body. Traditional treatment includesnon-steroidal anti-inflammatory drugs (NSAIDS), COX-2 inhibitors, anddisease-modifying anti-rheumatic drugs (DMARDS) such as methotrexate.None of the traditional treatment regimes is ideal, especially for longterm use.

Biological response modifiers, which target inflammatory mediators,offer a relatively new approach to the treatment of rheumatoid arthritisand other autoimmune diseases. Such biological response modifiersinclude antibodies, or active portions thereof, against variousinflammatory mediators such as IL-6, IL-6 receptor, TNF-alpha, IL-23 andIL-12.

Some of the first biological response modifiers were medicationstargeting tumor necrosis factor alpha (TNF-a), a pro-inflammatorycytokine involved in the pathogenesis of RA. Several anti-TNF-alphamedications are currently marketed for the treatment of RA. For example,Enbrel® (etanercept, Amgen) is a TNF-alpha blocker. Etanercept is adimeric fusion protein consisting of the extracellular ligand-bindingportion of the human 75 kilodalton (p75) tumor necrosis factor receptor(TNFR) linked to the Fe portion of human IgG1. The Fe component ofetanercept contains the CH2 domain, the CH3 domain and hinge region, butnot the CH1 domain of IgG1. Etanercept is produced in a Chinese hamsterovary (CHO) mammalian cell expression system. It consists of 934 aminoacids and an apparent molecular weight of about 150 kilodaltons. Enbrel®is ‘Used to treat rheumatoid arthritis, psoriatic arthritis, ankylosingspondylitis and plaque psoriasis. Serious side effects of Enbrel®include infections including tuberculosis, fungal infection, bacterialor viral infection due to opportunistic pathogens. Sepsis can alsooccur. Lymphoma, or other malignancies have also been reported.

Remicade® (infliximab) is a chimeric anti-TNF-alpha IgGk1 monoclonalantibody composed of human constant and murine variable regions.Remicade is administered by intravenous injection and is used to treatrheumatoid arthritis, psoriasis, Crohn's disease, ulcerative colitis,and ankylosing spondylitis. Side effects of Remicade include seriousinfection or sepsis, and rarely certain T-cell lymphomas. Other sideeffects include hepatotoxicity, certain severe hematologic events,hypersensitivity reactions and certain severe neurological events.

Other biological response modifiers include humanized anti-interleukin-6(IL-6) receptor antibodies. IL-6 is a cytokine that contributes toinflammation, swelling and joint damage in RA. One humanized anti-IL-6receptor antibody, Actemra (tocilizumab, Roche), is approved by the FDAand European Commission to treat adult patients with rheumatoidarthritis. Actemra is also approved in Japan for treatment of RA andjuvenile idiopathic arthritis (sJIA). Phase III studies showed thattreatment with Actemra as a monotherapy, or a combination with MTX orother DMARDs, reduced signs and symptoms of RA compared with othertherapies. Actemra is a humanized anti-human IL-6 receptor monoclonalantibody that competitively blocks the binding of iL-6 to its receptor.Thus, it inhibits the proliferative effects of iL-6, which lead tosynovial thickening and pannus formation in RA. Serious side effects ofActemra, include serious infections and hypersensitivity reactionsincluding a few cases of anaphylaxis. Other side effects include upperrespiratory tract infection, headache, nasopharyngitis, hypertension andincreased ALT.

Another common autoimmune disease is psoriasis. An overactive immunesystem can lead to high levels of iL-12 and IL-23, two cytokine proteinsthat have been found in psoriatic skin plaques. IL-12 and IL-23 areinvolved in inflammatory and immune responses such as natural killercell activation and CD4+ T-cell differentiation and activation.

One treatment for moderate or severe psoriasis involves subcutaneousinjection of Stelara™ (ustekinumab, Centocor Ortho Biotech, Inc.) ahumanized IgG1k monoclonal antibody against the p40 subunit of the IL-12and IL-23 cytokines. Stelara has been shown to provide relief fromcertain symptoms associated with psoriatic plaques, such as plaquethickness, scaling and redness. The formulation for Stelara includesL-histidine and L-histidine monohydrochloride monohydrate, polysorbate80, and sucrose in aqueous solution. Use of Stelara™ affects the immunesystem, and may increase chances of infection, including tuberculosis,and infections caused by bacteria, fungi or viruses; as well as increasethe risk of certain types of cancer.

Side effects of the biological response modifiers are significant andare caused in part by high levels following injection into patientsrenders patients susceptible to serious infection or death. This is amajor side effect associated with this important class of drugs. Onechallenge is avoiding the high initial level of activity from the doseof antibody required to provide a long treatment effect followinginjection.

In one embodiment, the disclosure provides a method to prepare aconditionally active biological response mediator, or fragment thereof,that avoids the high level of activity from the dose of antibodyrequired to provide a long treatment effect following injection. Themethod of the disclosure can be used to design antibodies toinflammatory mediators such as IL-6, IL-6 receptor, TNF-alpha, IL-23 andIL-12 that are inactive at dosing conditions such as room temperature,but slowly refold (reversibly or irreversibly) at body temperature.These antibodies or fragments thereof would be inactive upon initialinjection, but would refold or reactivate over a period of hours to dayswhen exposed to blood following injection. This could allow higherdosing, and a longer half-life (or periods between dosing) with reducedside effects.

In one aspect, the disclosure provides a method for preparation of aconditionally active antibody to an inflammatory mediator, or fragmentthereof, that is inactive at dosing conditions such as room temperature,but slowly refold (reversibly or irreversibly) at body temperature. Themethod comprises the following steps. Selecting an inflammatorymediator. Screening to identify an antibody to the inflammatory mediatorvia hybridoma. Humanizing the anti-inflammatory mediator antibody.Evolving the anti-inflammatory mediator antibody and screeningdifferentially for binding at two or more conditions, for example, twoor more temperature conditions such as at room temperature and at 37° C.or higher; selecting for mutations that are inactive at a firstcondition, relative to wild type, but show increased activity (e.g.binding) relative to the wild type antibody activity (binding) at asecond condition. The up-mutants identified in the heavy and lightchanges are then recombined within the heavy and light chains, as wellas through combinatorial association of the heavy and light chains.Screening of these recombined heavy and light chains is repeated at thetwo conditions, for example, room temperature and at 37° C. or higher.In addition, the recombined antibodies or fragments can be screened foractivity and stability under storage and physiological conditions.

Alternatively, the wild-type antibody to the inflammatory mediator is aknown antibody or variant or active fragment thereof.

In one aspect, the first and second conditions are selected fromconditions of pH, osmotic pressure, osmolality, oxidation andelectrolyte concentration. In another aspect, the inflammatory mediatoris selected from IL-6, IL-6 receptor, TNF-alpha, IL-23 and iL-12.

In another aspect, the disclosure provides a method for preparation of aconditionally active antibody to IL-6, or fragment thereof, that isinactive at dosing conditions such as room temperature, but slowlyrefold (reversibly or irreversibly) at body temperature. The methodcomprises the following steps. Screening a fully human library for anantibody to IL-6. Evolving the IL-6 antibody and screeningdifferentially for molecules at room temperature and at 37° C. orhigher; selecting for mutations that are inactive at room temperature,relative to wild type, but show increased activity (e.g. binding)relative to the wild type antibody activity (binding). The up-mutantsidentified in the heavy and light changes are then recombined within theheavy and light chains, as well as through combinatorial association ofthe heavy and light chains. Screening of these recombined heavy andlight chains is repeated at room temperature and the higher temperature.In addition, the recombined antibodies or fragments are tested foractivity and stability under storage and physiological conditions.

The conditionally active anti-IL-6 antibodies thus identified andproduced can be used in a method to treat an autoimmune disease, such asrheumatoid arthritis or psoriasis, by administration of an effectiveamount to a patient in need thereof, with a reduction in the severity ofside effects compared to administration of a traditional biologicalresponse modifier anti-IL-6 antibody. One advantage of this method isthat it allows for smoothing or leveling of the drug quantity over theperiod of treatment relative to the current high level of biologicalresponse modifier drug followed by half-life clearance over weeks ormonths.

One or more mutagenesis techniques are employed to evolve the DNA whichencodes the wild-type protein to create a library of mutant DNA; themutant DNA is expressed to create a library of mutant proteins; and thelibrary is subjected to a screening assay under a normal physiologicalcondition and under one or more aberrant conditions. Conditionallyactive biologic proteins are selected from those proteins which exhibitboth (a) a decrease in activity in the assay at the normal physiologicalcondition compared to the wild-type protein, and (b) an increase inactivity in the assay under the aberrant condition compared to thewild-type protein. Alternatively, conditionally active biologic proteinsare selected from those proteins which exhibit changes in activity,reversibly or irreversibly, in two or more different physiologicalconditions.

Generation of Evolved Molecules from Parent Molecule

Mirac Proteins can be generated through a process of mutagenesis andscreening for individual mutations for a reduction in activity at thewild-type condition with activity at non wild-type conditions remainingthe same or better than the activity at the wild-type condition.

The disclosure provides for a method for generating a nucleic acidvariant encoding a polypeptide having enzyme activity, wherein thevariant has an altered biological activity from that which naturallyoccurs, the method comprising (a) modifying the nucleic acid by (i)substituting one or more nucleotides for a different nucleotide, whereinthe nucleotide comprises a natural or non-natural nucleotide; (ii)deleting one or more nucleotides, (iii) adding one or more nucleotides,or (iv) any combination thereof. In one aspect, the non-naturalnucleotide comprises inosine. In another aspect, the method furthercomprises assaying the polypeptides encoded by the modified nucleicacids for altered enzyme activity, thereby identifying the modifiednucleic acid(s) encoding a polypeptide having altered enzyme activity.In one aspect, the modifications of step (a) are made by PCR,error-prone PCR, shuffling, oligonucleotide-directed mutagenesis,assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassettemutagenesis, recursive ensemble mutagenesis, exponential ensemblemutagenesis, site-specific mutagenesis, gene reassembly, gene sitesaturated mutagenesis, ligase chain reaction, in vitro mutagenesis,ligase chain reaction, oligonuclteotide synthesis, any DNA-generatingtechnique and any combination thereof. In another aspect, the methodfurther comprises at least one repetition of the modification step (a).

The disclosure further provides a method for making a polynucleotidefrom two or more nucleic acids, the method comprising: (a) identifyingregions of identity and regions of diversity between two or more nucleicacids, wherein at least one of the nucleic acids comprises a nucleicacid of the disclosure; (b) providing a set of oligonucleotides whichcorrespond in sequence to at least two of the two or more nucleic acids;and, (c) extending the oligonucleotides with a polymerase, therebymaking the polynucleotide.

Any technique of mutagenesis can be employed in various embodiments ofthe disclosure. Stochastic or random mutagenesis is exemplified by asituation in which a parent molecule is mutated (modified or changed) toyield a set of progeny molecules having mutation(s) that are notpredetermined. Thus, in an in vitro stochastic mutagenesis reaction, forexample, there is not a particular predetermined product whoseproduction is intended; rather there is an uncertainty—hencerandomness—regarding the exact nature of the mutations achieved, andthus also regarding the products generated. Stochastic mutagenesis ismanifested in processes such as error-prone PCR and stochasticshuffling, where the mutation(s) achieved are random or notpredetermined. The variant forms can be generated by error-pronetranscription, such as an error-prone PCR or use of a polymerase whichlacks proof-reading activity (see, Liao (1990) Gene 88:107-111), of thefirst variant form, or, by replication of the first form in a mutatorstrain (mutator host cells are discussed in further detail below, andare generally well known). A mutator strain can include any mutants inany organism impaired in the functions of mismatch repair. These includemutant gene products of mutS, mutT, mutH, mutL, ovrD, dcm, vsr, umuC,umuD, sbcB, red, etc. The impairment is achieved by genetic mutation,allelic replacement, selective inhibition by an added reagent such as asmall compound or an expressed antisense RNA, or other techniques.Impairment can be of the genes noted, or of homologous genes in anyorganism.

Current mutagenesis methods in widespread use for creating alternativeproteins from a starting molecule are oligonucleotide-directedmutagenesis technologies, error-prone polymerase chain reactions(error-prone PCR) and cassette mutagenesis, in which the specific regionto be optimized is replaced with a synthetically mutagenizedoligonucleotide. In these cases, a number of mutant sites are generatedaround certain sites in the original sequence.

In oligonucleotide-directed mutagenesis, a short sequence is replacedwith a synthetically mutagenized oligonucleotide. Inoligonucleotide-directed mutagenesis, a short sequence of thepolynucleotide is removed from the polynucleotide using restrictionenzyme digestion and is replaced with a synthetic polynucleotide inwhich various bases have been altered from the original sequence. Thepolynucleotide sequence can also be altered by chemical mutagenesis.Chemical mutagens include, for example, sodium bisulfite, nitrous acid,hydroxylamine, hydrazine or formic acid. Other agents which areanalogues of nucleotide precursors include nitrosoguanidine,5-bromouracil, 2-aminopurine, or acridine. Generally, these agents areadded to the PCR reaction in place of the nucleotide precursor therebymutating the sequence. Intercalating agents such as proflavine,acriflavine, quinacrine and the like can also be used. Randommutagenesis of the polynucleotide sequence can also be achieved byirradiation with X-rays or ultraviolet light. Generally, plasmidpolynucleotides so mutagenized are introduced into E. coli andpropagated as a pool or library of hybrid plasmids.

Error-prone PCR uses low-fidelity polymerization conditions to introducea low level of point mutations randomly over a long sequence. In amixture of fragments of unknown sequence, error-prone PCR can be used tomutagenize the mixture.

In cassette mutagenesis, a sequence block of a single template istypically replaced by a (partially) randomized sequence. Reidhaar-OlsonJ F and Sauer R T: Combinatorial cassette mutagenesis as a probe of theinformational content of protein sequences. Science 241(4861):53-57,1988.

Alternatively, any technique of non-stochastic or non-random mutagenesiscan be employed in various embodiments of the disclosure. Non-stochasticmutagenesis is exemplified by a situation in which a parent molecule ismutated (modified or changed) to yield a progeny molecule having one ormore predetermined mutations. It is appreciated that the presence ofbackground products in some quantity is a reality in many reactionswhere molecular processing occurs, and the presence of these backgroundproducts does not detract from the non-stochastic nature of amutagenesis process having a predetermined product. Site-saturationmutagenesis and synthetic ligation reassembly, are examples ofmutagenesis techniques where the exact chemical structure(s) of theintended product(s) are predetermined.

One method of site-saturation mutagenesis is disclosed in U.S. patentapplication publication 2009/0130718, which is incorporated herein byreference. This method provides a set of degenerate primerscorresponding to codons of a template polynucleotide, and performspolymerase elongation to produce progeny polynucleotides, which containsequences corresponding to the degenerate primers. The progenypolynucleotides can be expressed and screened for directed evolution.Specifically, this is a method for producing a set of progenypolynucleotides, comprising the steps of (a) providing copies of atemplate polynucleotide, each comprising a plurality of codons thatencode a template polypeptide sequence; and (b) for each codon of thetemplate polynucleotide, performing the steps of (1) providing a set ofdegenerate primers, where each primer comprises a degenerate codoncorresponding to the codon of the template polynucleotide and at leastone adjacent sequence that is homologous to a sequence adjacent to thecodon of the template polynucleotide; (2) providing conditions allowingthe primers to anneal to the copies of the template polynucleotides; and(3) performing a polymerase elongation reaction from the primers alongthe template; thereby producing progeny polynucleotides, each of whichcontains a sequence corresponding to the degenerate codon of theannealed primer; thereby producing a set of progeny polynucleotides.

Site-saturation mutagenesis relates to the directed evolution of nucleicacids and screening of clones containing the evolved nucleic acids forresultant activity(ies) of interest, such nucleic acid activity(ies)&/or specified protein, particularly enzyme, activity(ies) of interest.

Mutagenized molecules provided by this technique may have chimericmolecules and molecules with point mutations, including biologicalmolecules that contain a carbohydrate, a lipid, a nucleic acid, &/or aprotein component, and specific but non-limiting examples of theseinclude antibiotics, antibodies, enzymes, and steroidal andnon-steroidal hormones.

Site saturation mutagenesis relates generally to a method of: 1)preparing a progeny generation of molecule(s) (including a molecule thatis comprised of a polynucleotide sequence, a molecule that is comprisedof a polypeptide sequence, and a molecule that is comprised in part of apolynucleotide sequence and in part of a polypeptide sequence), that ismutagenized to achieve at least one point mutation, addition, deletion,&/or chimerization, from one or more ancestral or parental generationtemplate(s); 2) screening the progeny generation molecule(s)—preferablyusing a high throughput method—for at least one property of interest(such as an improvement in an enzyme activity or an increase instability or a novel chemotherapeutic effect); 3) optionally obtaining&/or cataloguing structural &/or and functional information regardingthe parental &/or progeny generation molecules; and 4) optionallyrepeating any of steps 1) to 3).

In site saturation mutagenesis, there is generated (e.g. from a parentpolynucleotide template)—in what is termed “codon site-saturationmutagenesis”—a progeny generation of polynucleotides, each having atleast one set of up to three contiguous point mutations (i.e. differentbases comprising a new codon), such that every codon (or every family ofdegenerate codons encoding the same amino acid) is represented at eachcodon position. Corresponding to—and encoded by—this progeny generationof polynucleotides, there is also generated a set of progenypolypeptides, each having at least one single amino acid point mutation.In a preferred aspect, there is generated—in what is termed “ino acidsite-saturation mutagenesis”—one such mutant polypeptide for each of the19 miturally encoded polypeptide-forming alpha-amino acid substitutionsat each and every amino acid position along the polypeptide. Thisyields—for each and every amino acid position along the parentalpolypeptide—a total of 20 distinct progeny polypeptides including theoriginal amino acid, or potentially more than 21 distinct progenypolypeptides if additional amino acids are used either instead of or inaddition to the 20 naturally encoded amino acids.

Other mutagenesis techniques can also be employed which involverecombination and more specifically a method for preparingpolynucleotides encoding a polypeptide by a method of in vivore-assortment of polynucleotide sequences containing regions of partialhomology, assembling the polynucleotides to form at least onepolynucleotide and screening the polynucleotides for the production ofpolypeptide(s) having a useful property.

In another aspect, mutagenesis techniques exploit the natural propertyof cells to recombine molecules and/or to mediate reductive processesthat reduce the complexity of sequences and extent of repeated orconsecutive sequences possessing regions of homology.

Various mutagenesis techniques can be used alone or in combination toprovide a method for generating hybrid polynucleotides encodingbiologically active hybrid polypeptides with enhanced activities. Inaccomplishing these and other objects, there has been provided, inaccordance with one aspect of the disclosure, a method for introducingpolynucleotides into a suitable host cell and growing the host cellunder conditions that produce a hybrid polynucleotide.

Chimeric genes have been made by joining 2 polynucleotide fragmentsusing compatible sticky ends generated by restriction enzyme(s), whereeach fragment is derived from a separate progenitor (or parental)molecule. Another example is the mutagenesis of a single codon position(i.e. to achieve a codon substitution, addition, or deletion) in aparental polynucleotide to generate a single progeny polynucleotideencoding for a single site-mutagenized polypeptide.

Further, in vivo site specific recombination systems have been utilizedto generate hybrids of genes, as well as random methods of in vivorecombination, and recombination between homologous but truncated geneson a plasmid. Mutagenesis has also been reported by overlappingextension and PCR.

Non-random methods have been used to achieve larger numbers of pointmutations and/or chimerizations, for example comprehensive or exhaustiveapproaches have been used to generate all the molecular species within aparticular grouping of mutations, for attributing functionality tospecific structural groups in a template molecule (e.g. a specificsingle amino acid position or a sequence comprised of two or more aminoacids positions), and for categorizing and comparing specific groupingof mutations.

Any of these or other methods of evolving can be employed in the presentdisclosure to generate a new population of molecules (library) from oneor more parent molecules.

Once formed, the constructs may, or may not be size fractionated on anagarose gel according to published protocols, inserted into a cloningvector, and transfected into an appropriate host cell.

Expression of Evolved Molecules

Once a library of mutant molecules is generated, DNA can be expressedusing routine molecular biology techniques. Thus, protein expression canbe directed using various known methods.

For example, briefly, a wild type gene can be evolved using any varietyof random or non-random methods such as those indicated herein. MutantDNA molecules are then digested and ligated into vector DNA, such asplasmid DNA using standard molecular biology techniques. Vector DNAcontaining individual mutants is transformed into bacteria or othercells using standard protocols. This can be done in an individual wellof a multi-well tray, such as a 96-well tray for high throughputexpression and screening. The process is repeated for each mutantmolecule.

Polynucleotides selected and isolated as described are introduced into asuitable host cell. A suitable host cell is any cell which is capable ofpromoting recombination and/or reductive reassortment. The selectedpolynucleotides are preferably already in a vector which includesappropriate control sequences. The host cell can be a higher eukaryoticcell, such as a mammalian cell, or a lower eukaryotic cell, such as ayeast cell, or preferably, the host cell can be a prokaryotic cell, suchas a bacterial cell. futroduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (e.g. Ecker and Davis, 1986, fuhibitionof gene expression in plant cells by expression of antisense RNA, ProcNatl Acad Sci USA, 83:5372-5376).

As representative examples of expression vectors which may be used,there may be mentioned viral particles, baculovirus, phage, plasmids,phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA(e.g., vaccinia, adenovirus, foul pox virus, pseudorabies andderivatives of SV40), PI-based artificial chromosomes, yeast plasmids,yeast artificial chromosomes, and any other vectors specific forspecific hosts of interest (such as bacillus, aspergillus and yeast).Thus, for example, the DNA may be included in any one of a variety ofexpression vectors for expressing a polypeptide. Such vectors includechromosomal, nonchromosomal and synthetic DNA sequences. Large numbersof suitable vectors are known to those of skill in the art, and arecommercially available. The following vectors are provided by way ofexample; Bacterial: pQE vectors (Qiagen), pBluescript plasmids, pNHvectors, (lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540,pRIT2T (Pharmacia); Eukaryotic: pXT1, pSGS (Stratagene), pSVK3, pBPV,pMSG, pSVLSV40 (Pharmacia). However, any other plasmid or other vectormay be used so long as they are replicable and viable in the host. Lowcopy number or high copy number vectors may be employed with the presentdisclosure.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct RNAsynthesis. Particular named bacterial promoters include lacI, lacZ, T3,T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include CMVimmediate early, HSV thymidine kinase, early and late SV40, LTRs fromretrovirus, and mouse metallothionein-1. Selection of the appropriatevector and promoter is well within the level of ordinary skill in theart. The expression vector also contains a ribosome binding site fortranslation initiation and a transcription terminator. The vector mayalso include appropriate sequences for amplifying expression. Promoterregions can be selected from any desired gene using chloramphenicoltransferase (CAT) vectors or other vectors with selectable markers. Inaddition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

Therefore, in another aspect of the disclosure, novel polynucleotidescan be generated by the process of reductive reassortment. The methodinvolves the generation of constructs containing consecutive sequences(original encoding sequences), their insertion into an appropriatevector, and their subsequent introduction into an appropriate host cell.The reassortment of the individual molecular identities occurs bycombinatorial processes between the consecutive sequences in theconstruct possessing regions of homology, or between quasi-repeatedunits. The reassortment process recombines and/or reduces the complexityand extent of the repeated sequences, and results in the production ofnovel molecular species. Various treatments may be applied to enhancethe rate of reassortment. These could include treatment withultra-violet light, or DNA damaging chemicals, and/or the use of hostcell lines displaying enhanced levels of “genetic instability”. Thus thereassortment process may involve homologous recombination or the naturalproperty of quasi-repeated sequences to direct their own evolution.

In one aspect, the host organism or cell comprises a gram negativebacterium, a gram positive bacterium or a eukaryotic organism. Inanother aspect of the disclosure, the gram negative bacterium comprisesEscherichia coli, or Pseudomonas fluorescens. In another aspect of thedisclosure, the gram positive bacterium comprise Streptomyces diversa,Lactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris, orBacillus subtilis. In another aspect of the disclosure, the eukaryoticorganism comprises Saccharomyces cerevisiae, Schizosaccharomyces pombe,Pichia pastoris, Kluyveromyces lactis, Hansenula plymorpha, orAspergillus niger. As representative examples of appropriate hosts,there may be mentioned: bacterial cells, such as E. coli, Streptomyces,Salmonella typhimurium; fungal cells, such as yeast; insect cells suchas Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS orBowes melanoma; adenoviruses; and plant cells. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

With particular references to various mammalian cell culture systemsthat can be employed to express recombinant protein, examples ofmammalian expression systems include the COS-7 lines of monkey kidneyfibroblasts, described in “SV40-transformed simian cells support thereplication of early SV40 mutants” (Gluzman, 1981), and other cell linescapable of expressing a compatible vector, for example, the C127, 3T3,CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprisean origin of replication, a suitable promoter and enhancer, and also anynecessary ribosome binding sites, polyadenylation site, splice donor andacceptor sites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The cells are then propagated and “reductive reassortment” is effected.The rate of the reductive reassortment process may be stimulated by theintroduction of DNA damage if desired. In vivo reassortment is focusedon “inter-molecular” processes collectively referred to as“recombination” which in bacteria, is generally viewed as a“RecA-dependent” phenomenon. The disclosure can rely on recombinationprocesses of a host cell to recombine and re-assort sequences, or thecells' ability to mediate reductive processes to decrease the complexityof quasi-repeated sequences in the cell by deletion. This processof“reductive reassortment” occurs by an “intra-molecular”,RecA-independent process. The end result is a reassortment of themolecules into all possible combinations.

Host cells containing the polynucleotides of interest can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying genes. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothe ordinarily skilled artisan.

Protein expression can be induced by a variety of known methods, andmany genetic systems have been published for induction of proteinexpression. For example, with appropriate systems, the addition of aninducing agent will induce protein expression. Cells are then pelletedby centrifugation and the supernatant removed. Periplasmic protein canbe enriched by incubating the cells with DNAse, RNAse, and lysozyme.After centrifugation, the supernatant, containing the new protein, istransferred to a new multi-well tray and stored prior to assay.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract is retained forfurther purification. Microbial cells employed for expression ofproteins can be disrupted by any convenient method, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents. Such methods are well known to those skilled in the art.The expressed polypeptide or fragment thereof can be recovered andpurified from recombinant cell cultures by methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Protein refolding steps can beused, as necessary, in completing configuration of the polypeptide. Ifdesired, high performance liquid chromatography (HPLC) can be employedfor final purification steps.

The clones which are identified as having the desired activity may thenbe sequenced to identify the polynucleotide sequence encoding an enzymehaving the enhanced activity.

The polypeptides that are identified from such libraries can be used fortherapeutic, diagnostic, research and related purposes, and/or can besubjected to one or more additional cycles of shuffling and/orselection. The disclosure provides for a fragment of the conditionallyactive biologic protein which is at least 10 amino acids in length, andwherein the fragment has activity.

The disclosure provides for a codon-optimized polypeptide or a fragmentthereof, having enzyme activity, wherein the codon usage is optimizedfor a particular organism or cell. Narum et al., “Codon optimization ofgene fragments encoding Plasmodium falciparum merzoite proteins enhancesDNA vaccine protein expression and immunogenicity in mice”. Infect.Immun. 2001 December, 69(12):7250-3 describes codon-optimization in themouse system. Outchkourov et al., “Optimization of the expression ofEquistatin in Pichia pastoris, protein expression and purification”,Protein Expr. Purif. 2002 February; 24(1):18-24 describescodon-optimization in the yeast system. Feng et al., “High levelexpression and mutagenesis of recombinant human phosphatidylcholinetransfer protein using a synthetic gene: evidence for a C-terminalmembrane binding domain” Biochemistry 2000 Dec. 19, 39(50):15399-409describes codon-optimization in E. coli. Humphreys et al., “High-levelperiplasmic expression in Escherichia coli using a eukaryotic signalpeptide: importance of codon usage at the 5′ end of the codingsequence”, Protein Expr. Purl£ 2000 Nov. 20(2):252-64 describes howcodon usage affects secretion in E. coli.

The evolution of a conditionally active biologic protein can be aided bythe availability of a convenient high throughput screening or selectionprocess.

Once identified, polypeptides and peptides of the disclosure can besynthetic, or be recombinantly generated polypeptides. Peptides andproteins can be recombinantly expressed in vitro or in vivo. Thepeptides and polypeptides of the disclosure can be made and isolatedusing any method known in the art. Polypeptide and peptides of thedisclosure can also be synthesized, whole or in part, using chemicalmethods well known in the art. See e.g., Caruthers (1980) “New chemicalmethods for synthesizing polynucleotides”, Nucleic Acids Res. Symp. Ser.215-223; Hom (1980), “Synthesis of oligonucleotides on cellulose. PartII: design and synthetic strategy to the synthesis of 22oligodeoxynucleotides coding for Gastric Inhibitory Polypeptide (GIP)I.,Nucleic Acids Res. Symp. Ser. 225-232; Banga, A. K., TherapeuticPeptides and Proteins, Formulation, Processing and Delivery Systems(1995) Technomic Publishing Co., Lancaster, Pa. For example, peptidesynthesis can be performed using various solid-phase techniques (seee.g., Roberge (1995) “A strategy for a convergent synthesis of N-linkedglycopeptides on a solid support”, Science 269:202; Merrifield (1997)“Concept and early development of solid-phase peptide synthesis”,Methods Enzymol. 289:3-13) and automated synthesis may be achieved,e.g., using the ABI 431A Peptide Synthesizer (Perkin Elmer) inaccordance with the instructions provided by the manufacturer.

The peptides and polypeptides of the disclosure can also beglycosylated. The glycosylation can be added post-translationally eitherchemically or by cellular biosynthetic mechanisms, wherein the latterincorporates the use of known glycosylation motifs, which can be nativeto the sequence or can be added as a peptide or added in the nucleicacid coding sequence. The glycosylation can be 0-link:ed or N-link:ed.

The peptides and polypeptides of the disclosure, as defined above,include all “mimetic” and “peptidomimetic” forms. The terms “mimetic”and “peptidomimetic” refer to a synthetic chemical compound which hassubstantially the same structural and/or functional characteristics ofthe polypeptides of the disclosure. The mimetic can be either entirelycomposed of synthetic, non-natural analogues of amino acids, or, is achimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The mimetic can also incorporate anyamount of natural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. As with polypeptides of the disclosure which areconservative variants, routine experimentation will determine whether amimetic is within the scope of the disclosure, i.e., that its structureand/or function is not substantially altered.

Polypeptide mimetic compositions of the disclosure can contain anycombination of non-natural structural components. In alternative aspect,mimetic compositions of the disclosure include one or all of thefollowing three structural groups: a) residue linkage groups other thanthe natural amide bond (“peptide bond”) linkages; b) non-naturalresidues in place of naturally occurring amino acid residues; or c)residues which induce secondary structural mimicry, i.e., to induce orstabilize a secondary structure, e.g., a beta turn, gamma turn, betasheet, alpha helix conformation, and the like. For example, apolypeptide of the disclosure can be characterized as a mimetic when allor some of its residues are joined by chemical means other than naturalpeptide bonds. Individual peptidomimetic residues can be joined bypeptide bonds, other chemical bonds or coupling means, such as, e.g.,glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(.dbd.O)—CH.sub.2- for —C(.dbd.O)—NH—), aminomethylene (CH.sub.2-NH),ethylene, olefin (CH.dbd.CH), ether (CH.sub.2-O), thioether(CH.sub.2-S), tetrazole (CN.sub.4-), thiazole, retroamide, thioamide, orester (see, e.g., Spatola (1983) in Chemistry and Biochemistry of AminoAcids, Peptides and Proteins, Vol. 7, pp 267.; 357, “Peptide BackboneModifications,” Marcell Dekker, N.Y.).

A polypeptide of the disclosure can also be characterized as a mimeticby containing all or some non-natural residues in place of naturallyoccurring amino acid residues. Non-natural residues are well describedin the scientific and patent literature; a few exemplary non-naturalcompositions useful as mimetics of natural amino acid residues andguidelines are described below. Mimetics of aromatic amino acids can begenerated by replacing by, e.g., D- or L-naphylalanine; D- orL-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2, 3-, or4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; D- orL-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,D- or L-alkylanines, where alkyl can be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of anon-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids can be generated by substitution by,e.g., non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′—N—C—N—R′) such as, e.g.,1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl orglutamyl can also be converted to asparaginyl and glutaminyl residues byreaction with ammonium ions. Mimetics of basic amino acids can begenerated by substitution with, e.g., (in addition to lysine andarginine) the amino acids ornithine, citrulline, or (guanidino)-aceticacid, or (guanidino)alkyl-acetic acid, where alkyl is defined above.Nitrile derivative (e.g., containing the CN-moiety in place of COOH) canbe substituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, e.g., one or more conventional reagents, including, e.g.,phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin,preferably under alkaline conditions. Tyrosine residue mimetics can begenerated by reacting tyrosyl with, e.g., aromatic diazonium compoundsor tetranitromethane. N-acetylimidizol and tetranitromethane can be usedto form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimeticscan be generated (and amino terminal residues can be altered) byreacting lysinyl with, e.g., succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitro-benzenesulfonic acid, 0-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate. Mimetics of methioninecan be generated by reaction with, e.g., methionine sulfoxide. Mimeticsof proline include, e.g., pipecolic acid, thiazolidine carboxylic acid,3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or3,3,-dimethylproline. Histidine residue mimetics can be generated byreacting histidyl with, e.g., diethylprocarbonate or para-bromophenacylbromide. Other mimetics include, e.g., those generated by hydroxylationof proline and lysine; phosphorylation of the hydroxyl groups of serylor threonyl residues; methylation of the alpha-amino groups of lysine,arginine and histidine; acetylation of the N-terminal amine; methylationof main chain amide residues or substitution with N-methyl amino acids;or amidation of C-terminal carboxyl groups.

A residue, e.g., an amino acid, of a polypeptide of the disclosure canalso be replaced by an amino acid (or peptidomimetic residue) of theopposite chirality. Thus, any amino acid naturally occurring in theL-configuration (which can also be referred to as the R or S, dependingupon the structure of the chemical entity) can be replaced with theamino acid of the same chemical structural type or a peptidomimetic, butof the opposite chirality, referred to as the D-amino acid, but also canbe referred to as the R- or S-form.

The disclosure also provides methods for modifying the polypeptides ofthe disclosure by either natural processes, such as post-translationalprocessing (e.g., phosphorylation, acylation, etc), or by chemicalmodification techniques. Modifications can occur anywhere in thepolypeptide, including the peptide backbone, the amino acid side-chainsand the amino or carboxyl termini. It will be appreciated that the sametype of modification may be present in the same or varying degrees atseveral sites in a given polypeptide. Also a given polypeptide may havemany types of modifications. Modifications include acetylation,acylation, PEGylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of a phosphatidylinositol,cross-linking cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristolyation, oxidation, pegylation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,and transfer-RNA mediated addition of amino acids to protein such asarginylation. See, e.g., Creighton, T. E., Proteins—Structure andMolecular Properties 2nd Ed., W. H. Freeman and Company, New York(1993); Posttranslational Covalent Modification of Proteins, B. C.Johnson, Ed., Academic Press, New York pp. 1-12 (1983).

Solid-phase chemical peptide synthesis methods can also be used tosynthesize the polypeptide or fragments of the disclosure. Such methodshave been known in the art since the early 1960's (Merrifield, R. B.,“Solid-phase synthesis.!. The synthesis of a tetrapeptide”, J. Am. Chem.Soc., 85:2149-2154, 1963) (See also Stewart, J. M. and Young, J. D.,Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford,Ill., pp. 11-12)) and have recently been employed in commerciallyavailable laboratory peptide design and synthesis kits (CambridgeResearch Biochemicals). Such commercially available laboratory kits havegenerally utilized the teachings of H. M. Geysen et al., “Use of peptidesynthesis to probe viral antigens for epitopes to a resolution of asingle amino acid,” Proc. Natl. Acad. Sci., USA, 81:3998 (1984) andprovide for synthesizing peptides upon the tips of a multitude of “rods”or “pins” all of which are connected to a single plate. When such asystem is utilized, a plate of rods or pins is inverted and insertedinto a second plate of corresponding wells or reservoirs, which containsolutions for attaching or anchoring an appropriate amino acid to thepin's or rod's tips. By repeating such a process step, i.e., invertingand inserting the rod's and pin's tips into appropriate solutions, aminoacids are built into desired peptides. In addition, a number ofavailable FMOC peptide synthesis systems are available. For example,assembly of a polypeptide or fragment can be carried out on a solidsupport using an Applied Biosystems, Inc. Model 431A™ automated peptidesynthesizer. Such equipment provides ready access to the peptides of thedisclosure, either by direct synthesis or by synthesis of a series offragments that can be coupled using other known techniques.

The synthetic polypeptide or fragment thereof can be recovered andpurified by known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the polypeptide. If desired, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The disclosure provides for a conditionally active protein variantpreparation or formulation which comprises at least one of the proteinvariants, wherein the preparation is liquid or dry. The proteinformulation optionally includes a buffer, cofactor, second or additionalprotein, or one or more excipients. In one aspect the formulation isutilized as a therapeutic conditionally active biologic protein which isactive under aberrant or non-physiological conditions, but less activeor inactive under normal physiological conditions of, e.g., temperature,pH, or osmotic pressure, oxidation or osmolality.

Standard purification techniques can be employed for either recombinantor synthetic conditionally active biologic proteins.

Screening of Mutants to Identify Reversible or Non-Reversible Mutants

Identifying desirable molecules is most directly accomplished bymeasuring protein activity at the permissive condition and the wild typecondition. The mutants with the largest ratio of activity(permissive/wild type) can then be selected and permutations of thepoint mutations are generated by combining the individual mutationsusing standard methods. The combined permutation protein library is thenscreened for those proteins displaying the largest differential activitybetween the permissive and wild type condition.

Activity of supernatants can be screened using a variety of methods, forexample using high throughput activity assays, such as fluorescenceassays, to identify protein mutants that are sensitive at whatevercharacteristic one desires (temperature, pH, etc). For example, toscreen for temporally sensitive mutants, the enzymatic or antibodyactivity of each individual mutant is determined at lower temperatures(such as 25 degrees Celsius), and at temperatures which the originalprotein functions (such as 37 degrees Celsius), using commerciallyavailable substrates. Reactions can initially be performed in a multiwell assay format, such as a 96-well assay, and confirmed using adifferent format, such as a 14 ml tube format.

The disclosure further provides a screening assay for identifying aenzyme, the assay comprising: (a) providing a plurality of nucleic acidsor polypeptides; (b) obtaining polypeptide candidates to be tested forenzyme activity from the plurality; (c) testing the candidates forenzyme activity; and (d) identifying those polypeptide candidates whichexhibit elevated enzyme activity under aberrant or non-physiologicalconditions, and decreased enzyme activity compared to the wild-typeenzyme protein under normal physiological conditions of, e.g.,temperature, pH, oxidation, osmolality, electrolyte concentration orosmotic pressure.

In one aspect, the method further comprises modifying at least one ofthe nucleic acids or polypeptides prior to testing the candidates forconditional biologic activity. In another aspect, the testing of step(c) further comprises testing for improved expression of the polypeptidein a host cell or host organism. In a further aspect, the testing ofstep (c) further comprises testing for enzyme activity within a pH rangefrom about pH 3 to about pH 12. In a further aspect, the testing of step(c) further comprises testing for enzyme activity within a pH range fromabout pH 5 to about pH 10. In a further aspect, the testing of step (c)further comprises testing for enzyme activity within a pH range fromabout pH 6 to about pH 8. In a further aspect, the testing of step (c)further comprises testing for enzyme activity at pH 6.7 and pH 7.5. Inanother aspect, the testing of step (c) further comprises testing forenzyme activity within a temperature range from about 4 degrees C. toabout 55 degrees C. In another aspect, the testing of step (c) furthercomprises testing for enzyme activity within a temperature range fromabout 15 degrees C. to about 47 degrees C. In another aspect, thetesting of step (c) further comprises testing for enzyme activity withina temperature range from about 20 degrees C. to about 40 degrees C. Inanother aspect, the testing of step (c) further comprises testing forenzyme activity at the temperatures of 25 degrees C. and 37 degrees C.In another aspect, the testing of step (c) further comprises testing forenzyme activity under normal osmotic pressure, and aberrant (positive ornegative) osmotic pressure. In another aspect, the testing of step (c)further comprises testing for enzyme activity under normal electrolyteconcentration, and aberrant (positive or negative) electrolyteconcentration. The electrolyte concentration to be tested is selectedfrom one of calcium, sodium, potassium, magnesium, chloride, bicarbonateand phosphate concentration. In another aspect, the testing of step (c)further comprises testing for enzyme activity which results in astabilized reaction product.

In another aspect, the disclosure provides for a purified antibody thatspecifically binds to the polypeptide of the disclosure or a fragmentthereof, having enzyme activity. In one aspect, the disclosure providesfor a fragment of the antibody that specifically binds to a polypeptidehaving enzyme activity.

Antibodies and Antibody-Based Screening Methods

The disclosure provides isolated or recombinant antibodies thatspecifically bind to an enzyme of the disclosure. These antibodies canbe used to isolate, identify or quantify the enzymes of the disclosureor related polypeptides. These antibodies can be used to isolate otherpolypeptides within the scope the disclosure or other related enzymes.The antibodies can be designed to bind to an active site of an enzyme.Thus, the disclosure provides methods of inhibiting enzymes using theantibodies of the disclosure.

The antibodies can be used in immunoprecipitation, staining,immunoaffinity columns, and the like. If desired, nucleic acid sequencesencoding for specific antigens can be generated by immunization followedby isolation of polypeptide or nucleic acid, amplification or cloningand immobilization of polypeptide onto an array of the disclosure.Alternatively, the methods of the disclosure can be used to modify thestructure of an antibody produced by a cell to be modified, e.g., anantibody's affinity can be increased or decreased. Furthermore, theability to make or modify antibodies can be a phenotype engineered intoa cell by the methods of the disclosure.

Methods of immunization, producing and isolating antibodies (polyclonaland monoclonal) are known to those of skill in the art and described inthe scientific and patent literature, see, e.g., Coligan, CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991); Stites (eds.) BASICAND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos,Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975)“Continuous cultures of fused cells secreting antibody of predefinedspecificity”, Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORYMANUAL, Cold Spring Harbor Publications, New York. Antibodies also canbe generated in vitro, e.g., using recombinant antibody binding siteexpressing phage display libraries, in addition to the traditional invivo methods using animals. See, e.g., Hoogenboom (1997) “Designing andoptimizing library selection strategies for generating high-affinityantibodies”, Trends Biotechnol. 15:62-70; and Katz (1997) “Structuraland mechanistic determinants of affinity and specificity of ligandsdiscovered or engineered by phage display”, Annu. Rev. Biophys. Biomol.Struct. 26:27-45.

Polypeptides or peptides can be used to generate antibodies which bindspecifically to the polypeptides, e.g., the enzymes, of the disclosure.The resulting antibodies may be used in immunoaffinity chromatographyprocedures to isolate or purify the polypeptide or to determine whetherthe polypeptide is present in a biological sample. In such procedures, aprotein preparation, such as an extract, or a biological sample iscontacted with an antibody capable of specifically binding to one of thepolypeptides of the disclosure.

In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or other column matrix. The protein preparationis placed in contact with the antibody under conditions in which theantibody specifically binds to one of the polypeptides of thedisclosure. After a wash to remove non-specifically bound proteins, thespecifically bound polypeptides are eluted.

The ability of proteins in a biological sample to bind to the antibodymay be determined using any of a variety of procedures familiar to thoseskilled in the art. For example, binding may be determined by labelingthe antibody with a detectable label such as a fluorescent agent, anenzymatic label, or a radioisotope. Alternatively, binding of theantibody to the sample may be detected using a secondary antibody havingsuch a detectable label thereon. Particular assays include ELISA assays,sandwich assays, radioimmunoas says, and Western Blots.

Polyclonal antibodies generated against the polypeptides of thedisclosure can be obtained by direct injection of the polypeptides intoan animal or by administering the polypeptides to a non-human animal.The antibody so obtained will then bind the polypeptide itself. In thismanner, even a sequence encoding only a fragment of the polypeptide canbe used to generate antibodies which may bind to the whole nativepolypeptide. Such antibodies can then be used to isolate the polypeptidefrom cells expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique, the trioma technique, thehuman B-cell hybridoma technique, and the EBV-hybridoma technique (see,e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (see,e.g., U.S. Pat. No. 4,946,778) can be adapted to produce single chainantibodies to the polypeptides of the disclosure. Alternatively,transgenic mice maybe used to express humanized antibodies to thesepolypeptides or fragments thereof. Antibodies generated against thepolypeptides of the disclosure may be used in screening for similarpolypeptides (e.g., enzymes) from other organisms and samples. In suchtechniques, polypeptides from the organism are contacted with theantibody and those polypeptides which specifically bind the antibody aredetected. Any of the procedures described above may be used to detectantibody binding.

Screening Methodologies and “on-Line” Monitoring Devices

In practicing the methods of the disclosure, a variety of apparatus andmethodologies can be used to in conjunction with the polypeptides andnucleic acids of the disclosure, e.g., to screen polypeptides for enzymeactivity, to screen compounds as potential modulators, e.g., activatorsor inhibitors, of an enzyme activity, for antibodies that bind to apolypeptide of the disclosure, for nucleic acids that hybridize to anucleic acid of the disclosure, to screen for cells expressing apolypeptide of the disclosure and the like.

Arrays, or “Biochips”

Nucleic acids or polypeptides of the disclosure can be immobilized to orapplied to an array. Arrays can be used to screen for or monitorlibraries of compositions (e.g., small molecules, antibodies, nucleicacids, etc.) for their ability to bind to or modulate the activity of anucleic acid or a polypeptide of the disclosure. For example, in oneaspect of the disclosure, a monitored parameter is transcript expressionof an enzyme gene. One or more, or, all the transcripts of a cell can bemeasured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array, or “biochip.” By using an “array” of nucleic acids on amicrochip, some or all of the transcripts of a cell can besimultaneously quantified. Alternatively, arrays comprising genomicnucleic acid can also be used to determine the genotype of a newlyengineered strain made by the methods of the disclosure. Polypeptidearrays” can also be used to simultaneously quantify a plurality ofproteins. The present disclosure can be practiced with any known“array,” also referred to as a “microarray” or “nucleic acid array” or“polypeptide array” or “antibody array” or “biochip,” or variationthereof. Arrays are generically a plurality of “spots” or “targetelements,” each target element comprising a defined amount of one ormore biological molecules, e.g., oligonucleotides, immobilized onto adefined area of a substrate surface for specific binding to a samplemolecule, e.g., mRNA transcripts.

In practicing the methods of the disclosure, any known array and/ormethod of making and using arrays can be incorporated in whole or inpart, or variations thereof, as described, for example, in U.S. Pat.Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695;6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174;5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522;5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g.,WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g.,Johnston (1998) “Gene chips: Array of hope for understanding generegulation”, Curr. Bioi. 8:R171-R174; Schummer (1997) “InexpensiveHandheld Device for the Construction of High-Density Nucleic AcidArrays”, Biotechniques 23:1087-1092; Kern (1997) “Direct hybridizationof large-insert genomic clones on high-density gridded cDNA filterarrays”, Biotechniques 23:120-124; Solinas-Toldo (1997) “Matrix-BasedComparative Genomic Hybridization: Biochips to Screen for Genomichnbalances”, Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999)“Options Available—From Start to Finish—for Obtaining Expression Data byMicroarray”, Nature Genetics Supp. 21:25-32. See also published U.S.patent applications Nos. 20010018642; 20010019827; 20010016322;20010014449; 20010014448; 20010012537; 20010008765.

Capillary Arrays

Capillary arrays, such as the GIGAMATR.JX™ Diversa Corporation, SanDiego, Calif., can be used in the methods of the disclosure. Nucleicacids or polypeptides of the disclosure can be immobilized to or appliedto an array, including capillary arrays. Arrays can be used to screenfor or monitor libraries of compositions (e.g., small molecules,antibodies, nucleic acids, etc.) for their ability to bind to ormodulate the activity of a nucleic acid or a polypeptide of thedisclosure. Capillary arrays provide another system for holding andscreening samples. For example, a sample screening apparatus can includea plurality of capillaries formed into an array of adjacent capillaries,wherein each capillary comprises at least one wall defining a lumen forretaining a sample. The apparatus can further include interstitialmaterial disposed between adjacent capillaries in the array, and one ormore reference indicia formed within of the interstitial materiaL Acapillary for screening a sample, wherein the capillary is adapted forbeing bound in an array of capillaries, can include a first walldefining a lumen for retaining the sample, and a second wall formed of afiltering material, for filtering excitation energy provided to thelumen to excite the sample. A polypeptide or nucleic acid, e.g., aligand, can be introduced into a first component into at least a portionof a capillary of a capillary array. Each capillary of the capillaryarray can comprise at least one wall defining a lumen for retaining thefirst component. An air bubble can be introduced into the capillarybehind the first component. A second component can be introduced intothe capillary, wherein the second component is separated from the firstcomponent by the air bubble. A sample of interest can be introduced as afirst liquid labeled with a detectable particle into a capillary of acapillary array, wherein each capillary of the capillary array comprisesat least one wall defining a lumen for retaining the first liquid andthe detectable particle, and wherein the at least one wall is coatedwith a binding material for binding the detectable particle to the atleast one wall. The method can further include removing the first liquidfrom the capillary tube, wherein the bound detectable particle ismaintained within the capillary, and introducing a second liquid intothe capillary tube. The capillary array can include a plurality ofindividual capillaries comprising at least one outer wall defining alumen. The outer wall of the capillary can be one or more walls fusedtogether. Similarly, the wall can define a lumen that is cylindrical,square, hexagonal or any other geometric shape so long as the walls forma lumen for retention of a liquid or sample. The capillaries of thecapillary array can be held together in close proximity to form a planarstructure. The capillaries can be bound together, by being fused (e.g.,where the capillaries are made of glass), glued, bonded, or clampedside-by-side. The capillary array can be formed of any number ofindividual capillaries, for example, a range from 100 to 4,000,000capillaries. A capillary array can form a micro titer plate having about100,000 or more individual capillaries bound together.

Pharmaceutical Compositions

The present disclosure provides at least one composition comprising (a)a conditionally active biologic protein; and (b) a suitable carrier ordiluent. The present disclosure also provides at least one compositioncomprising (a) a conditionally active biologic protein encoding nucleicacid as described herein; and (b) a suitable carrier or diluent. Thecarrier or diluent can optionally be pharmaceutically acceptable,according to known carriers or diluents. The composition can optionallyfurther comprise at least one further compound, protein or composition.

The conditionally active biologic protein may be in the form of apharmaceutically acceptable salt. Pharmaceutically acceptable saltsmeans which can be generally used as salts of an therapeutic protein inpharmaceutical industry, including for example, salts of sodium,potassium, calcium and the like, and amine salts of procaine,dibenzylamine, ethylenediamine, ethanolamine, methylglucamine, taurine,and the like, as well as acid addition salts such as hydrochlorides, andbasic amino acids and the like.

The present disclosure further provides at least one conditionallyactive biologic protein method or composition, for administering atherapeutically effective amount to modulate or treat at least oneparent molecule related condition in a cell, tissue, organ, animal orpatient and/or, prior to, subsequent to, or during a related condition,as known in the art and/or as described herein. Thus, the disclosureprovides a method for diagnosing or treating a condition associated withthe wild-type protein in a cell, tissue, organ or animal, comprisingcontacting or administering a composition comprising an effective amountof at least one conditionally active biologic protein of the disclosurewith, or to, the cell, tissue, organ or animal. The method canoptionally further comprise using an effective amount of 0.001-50mg/kilogram of a conditionally active biologic protein of the disclosureto the cells, tissue, organ or animal. The method can optionally furthercomprise using the contacting or the administrating by at least one modeselected from parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracelebellar,intracerebroventricular, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal. Themethod can optionally further comprise administering, prior,concurrently, or after the conditionally active biologic proteincontacting or administering at least one composition comprising aneffective amount of at least one compound or protein selected from atleast one of a detectable label or reporter, a TNF antagonist, anantirheumatic, a muscle relaxant, a narcotic, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anesthetic, a neuromuscular blocker, an antimicrobial, anantipsoriatic, a corticosteriod, an anabolic steroid, an erythropoietin,an immunization, an immunoglobulin, an immunosuppressive, a growthhormone, a hormone replacement drug, a radiopharmaceutical, anantidepressant, an antipsychotic, a stimulant, an asthma medication, abeta agonist, an inhaled steroid, an epinephrine or analog thereof, acytotoxic or other anti-cancer agent, an anti-metabolite such asmethotrexate, or an anti-proliferative agent.

The present disclosure further provides at least one conditionallyactive biologic protein method for diagnosing at least one wild-typeprotein related condition in a cell, tissue, organ, animal or patientand/or, prior to, subsequent to, or during a related condition, as knownin the art and/or as described herein.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention. A variety of aqueous carriers can be used, e.g.,buffered saline and the like. These solutions are sterile and generallyfree of undesirable matter. These compositions may be sterilized byconventional, well known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like. The concentration of conditionally active biologicprotein in these formulations can vary widely, and will be selectedprimarily based on fluid volumes, viscosities, body weight and the likein accordance with the particular mode of administration selected andthe patient's needs.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the packaged nucleic acidsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Pharmaceuticalcompositions and formulations of the invention for oral administrationcan be formulated using pharmaceutically acceptable carriers well knownin the art in appropriate and suitable dosages. Such carriers enable thepharmaceuticals to be formulated in unit dosage forms as tablets, pills,powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries,suspensions, etc., suitable for ingestion by the patient. Pharmaceuticalpreparations for oral use can be formulated as a solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable additional compounds, if desired, toobtain tablets or dragee cores. Suitable solid excipients arecarbohydrate or protein fillers include, e.g., sugars, includinglactose, sucrose, mannitol, or sorbitol; starch from com, wheat, rice,potato, or other plants; cellulose such as methyl cellulose,hydroxypropylmethyl cellulose, or sodium carboxy-methylcellulose; andgums including arabic and tragacanth; and proteins, e.g., gelatin andcollagen. Disintegrating or solubilizing agents may be added, such asthe cross-linked polyvinylpyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate. Tablet forms can include one or moreof lactose, sucrose, mannitol, sorbitol, calcium phosphates, com starch,potato starch, tragacanth, microcrystalline cellulose, acacia, gelatin,colloidal silicon dioxide, croscannellose sodium, talc, magnesiumstearate, stearic acid, and other excipients, colorants, fillers,binders, diluents, buffering agents, moistening agents, preservatives,flavoring agents, dyes, disintegrating agents, and pharmaceuticallyacceptable carriers.

The invention provides aqueous suspensions comprising a conditionallyactive biologic protein, in admixture with excipients suitable for themanufacture of aqueous suspensions. Such excipients include a suspendingagent, such as sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia, and dispersing or wetting agents such as anaturally occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethylene oxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensationproduct of ethylene oxide with a partial ester derived from fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate).The aqueous suspension can also contain one or more preservatives suchas ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

Lozenge forms can comprise the active ingredient in a flavor, usuallysucrose and acacia or tragacanth, as well as pastilles comprising theactive ingredient in an inert base, such as gelatin and glycerin orsucrose and acacia emulsions, gels, and the like containing, in additionto the active ingredient, carriers known in the art. It is recognizedthat the conditionally active biologic protein, when administeredorally, must be protected from digestion. This is typically accomplishedeither by complexing the conditionally active biologic protein with acomposition to render it resistant to acidic and enzymatic hydrolysis orby packaging the conditionally active biologic protein in anappropriately resistant carrier such as a liposome. Means of protectingproteins from digestion are well known in the art. The pharmaceuticalcompositions can be encapsulated, e.g., in liposomes, or in aformulation that provides for slow release of the active ingredient.

The packaged conditionally active biologic protein, alone or incombination with other suitable components, can be made into aerosolformulations (e.g., they can be “nebulized”) to be administered viainhalation. Aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like. Suitable formulations for rectal administrationinclude, for example, suppositories, which consist of the packagednucleic acid with a suppository base. Suitable suppository bases includenatural or synthetic triglycerides or paraffin hydrocarbons. Inaddition, it is also possible to use gelatin rectal capsules whichconsist of a combination of the packaged nucleic acid with a base,including, for example, liquid triglycerides, polyethylene glycols, andparaffin hydrocarbons.

Dermal or topical delivery compositions of the invention may include inaddition to a conditionally active biologic protein, a pharmaceuticallyacceptable carrier in a cream, ointment, solution or hydrogelformulation, and other compounds so long as the added component does notdeleteriously affect delivery of the therapeutic protein. Conventionalpharmaceutically acceptable emulsifiers, surfactants, suspending agents,antioxidants, osmotic enhancers, extenders, diluents and preservativesmay also be added. Water soluble polymers can also be used as carriers.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally. In one aspect, parenteral modes ofadministration are preferred methods of administration for compositionscomprising a conditionally active biologic protein. The compositions mayconveniently be administered in unit dosage form and may be prepared byany of the methods well-known in the pharmaceutical art, for example asdescribed in Remington's Pharmaceutical Sciences, Mack Publishing Co.Easton Pa., 18th Ed., 1990. Formulations for intravenous administrationmay contain a pharmaceutically acceptable carrier such as sterile wateror saline, polyalkylene glycols such as polyethylene glycol, oils ofvegetable origin, hydrogenated naphthalenes and the like. Also see andadapt the description in U.S. Pat. No. 4,318,905.

The formulations of packaged compositions comprising a conditionallyactive biologic protein can be presented in unit-dose or multi-dosesealed containers, such as ampoules and vials. Injection solutions andsuspepsions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

The present disclosure also provides at least one conditionally activebiologic protein composition, device and/or method of delivery fordiagnosing of at least one wild-type protein related condition,according to the present disclosure.

Also provided is a composition comprising at least one conditionallyactive biologic protein and at least one pharmaceutically acceptablecarrier or diluent. The composition can optionally further comprise aneffective amount of at least one compound or protein selected from atleast one of a detectable label or reporter, a cytotoxic or otheranti-cancer agent, an anti-metabolite such as methotrexate, ananti-proliferative agent, a cytokine, or a cytokine antagonist, a TNFantagonist, an antirheumatic, a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic,a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an epinephrineor analog.

Also provided is a medical device, comprising at least one conditionallyactive biologic protein of the disclosure, wherein the device issuitable to contacting or administering the at least one conditionallyactive biologic protein by at least one mode selected from parenteral,subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracelebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.

In a further aspect, the disclosure provides a kit comprising at leastone conditionally active biologic protein or fragment of the disclosurein lyophilized form in a first container, and an optional secondcontainer comprising sterile water, sterile buffered water, or at leastone preservative selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuricnitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesiumchloride, alkylparaben, benzalkonium chloride, benzethonium chloride,sodium dehydroacetate and thimerosal, or mixtures thereof in an aqueousdiluent. In one aspect, in the kit, the concentration of conditionallyactive biologic protein or specified portion or variant in the firstcontainer is reconstituted to a concentration of about 0.1 mg/ml toabout 500 mg/ml with the contents of the second container. In anotheraspect, the second container further comprises an isotonicity agent. Inanother aspect, the second container further comprises a physiologicallyacceptable buffer. In one aspect, the disclosure provides a method oftreating at least one wild-type protein mediated condition, comprisingadministering to a patient in need thereof a formulation provided in akit and reconstituted prior to administration.

Also provided is an article of manufacture for human pharmaceutical ordiagnostic use, comprising packaging material and a container comprisinga solution or a lyophilized form of at least one conditionally activebiologic protein of the present disclosure. The article of manufacturecan optionally comprise having the container as a component of aparenteral, subcutaneous, intramuscular, intravenous, intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracelebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, ortransdermal delivery device or system.

The present disclosure further provides any disclosure described herein.

Example 1 General Description of a Multiwall Assay (for Example, 96-WellAssay) for Temperature Mutants

Fluorescent substrate is added to each well of a multiwall plate, atboth wild-type and new, lower reaction temperatures (for example, either37° C. or 25° C. as mentioned above) for an appropriate time period.Fluorescence is detected by measuring fluorescence in a fluorescentplate reader at appropriate excitation and emission spectra (forexample, 320 nm exitation/405 nm emission). Relative fluorescence units(RFU) are determined. Supernatant from wild type molecule andplasmid/vector transformed cells are used as positive and negativecontrols. Duplicate reactions are performed for each sample, reactiontemperature; and positive and negative control.

Mutants that are active at the lower temperature (for example, themutants active at 25° C.) and that have a decrease in activity at thewild type temperature (for example, a 10%, 20%, 30%, 40% or moredecrease in activity at 37° C.), thus having a ratio of activitiesgreater than or equal to about 1.1 or more (e.g., the ratio of theactivities at 25° C. or 37° C. (25° C./37° C.) is greater than or equalto 1.1 or more), can be deemed to be putative primary temperaturesensitive hits. These putative primary temperature sensitive hits canthen be rescreened, using the same assay, to confirm any primary hits.

Example 2 General Description of a Different Assay Format forConfirmation of Activity (for Example, a 14-mL Assay) for TemperatureMutants

Mutants that are identified as temperature sensitive primary hits areexpressed in 14 ml culture tubes and their enzymatic activity ismeasured at wild type (for example, 37° C.) and the lower temperature(for example, 25° C.). Protein is expressed and purified as describedabove for the multiwall format, with the exception that the expressionis performed in different format (14 ml tubes) rather than the multiwall(96-well plate) format.

Each mutant supernatant is transferred to a multiwall plate, for examplea 96-well microplate. Fluorescent substrate is added to each tube at theindicated reaction temperatures (wild-type, lower temperature) for arequired period of time. Wild-type molecules are used as a positivecontrol and supernatant from cells transformed with only vector is usedas a negative control. Fluorescence is detected by measuringfluorescence in a fluorescent plate reader at the appropriate emissionspectra (for example, 320 nm exitation/405 nm emission). Relativefluorescence units (RFU) are determined. Duplicate reactions can areperformed for each sample, reaction temperature, and positive andnegative control.

Mutants that are active at the lower temperatures (for example, 25° C.)but that demonstrate at least a 30% or more decreased activity at wildtype (for example, 37° C.), thus have a ratio of activity at lowertemperature (for example, 25° C.) to wild type temperature (for example,37° C.) equal to or greater than 1.5, are identified as temperaturesensitive hits.

The activities of mutants at the lower temperature (for example 25° C.)are compared to the activity of the wild-type molecule at the wild-typetemperature (for example 37° C.). If molecules are more active than thewild-type molecules at the lower temperature (for example 25° C.), asindicated by a residual activity>1′ preferably 2 or greater than 2, andif the mut ts demonstrate an overall decrease in activity when comparedto the wild-type molecule at the wild-type temperature (37° C.), thephenotype of the mutants as temperature sensitive mutants can beconfirmed.

Example 3 General Description of Further Evolution of Hits Discovered

If desired, a new, combinatorial variant library is generated from allor selected mutant hits previously identified. The new library can bedesigned to contain every possible combination of amino acid variantsfor each of the selected mutants, and rescreened as described for newhits.

Example 4 General Description of Reversibility of Enzymatic ActivityFollowing Decrease in Temperature

Temperature sensitive, evolved mutants can be further assayed todetermine whether enzymatic activity at lower temperatures (for example,25° C.) is reversible or irreversible by exposing the mutants toelevated temperatures followed by a return to the lower temperature (forexample, 25° C.). The temperature sensitive mutants are expressed in anydesired format, for example in 14 ml culture tubes, as brieflydescribed. The mutants are tested for their activities under severalconditions, including the wild-type temperature (for example, 37° C.) aswell as other temperatures, and subsequently re-exposure to therequisite lower temperature of (25° C. for example). Mutants that areactive at lower temperatures, show decreased activity when raised tohigher or wild-type temperatures (i.e., the ratio of the activities atlower to higher temperatures is equal to or greater than 1, 1.5, or 2 orhigher), and exhibit a baseline activity when lowered again to the lowertemperature are scored as “Reversible Hits”. Mutants that are active atthe lower temperature, show decreased activity when raised to higher orwild-type temperatures (i.e., the ratio of the activities at the lowerto higher temperatures is equal to or greater than 1, 1.5 or 2 orhigher), and exhibit at least the same amount of decreased activity whenlowered again to the lower temperature are scored as “IrreversibleHits”.

Example 5 Materials and Methods to Screen for Conditionally ActiveAngiostatin Variants

Materials and methods to screen for conditionally active angiostatinvariants can be adapted from Chi and Pizzo, “Angiosatin is directlycytotoxic to tumor cells at low extracellular pH: a mechanism dependenton cell surface-associated ATP synthase”, Cancer Res. 2006;66(2):875-882, which is incorporated herein by reference.

Materials. Wild-type angiostatin kringles 1 to 3, derived from humanplasminogen, can be obtained from Calbiochem (Darmstadt, Germany) andreconstituted in sterile PBS. Polyclonal antibodies directed against thecatalytic beta-subunit of ATP synthase can be generated and bovine F1ATP synthase subunit can be purified as previously described (Moser etal., “Angiostatin binds ATP synthase on the surface of human endothelialcells”, Proc Natl Acad Sci US A 1999; 96:2811-6; Moser et al.“Endothelial cell surface F1-FO ATP synthase is active in ATP synthesisand is inhibited by angiostatin”, Proc Natl Acad Sci USA; 2001;98:6656-61). Cariporide can be solubilized in sterile water and sterilefiltered.

Cell culture. A549 (human epithelial cell line derived from a lungcarcinoma tissue), or an alternative cancer cell line (DU145, LNCaP, orPC-3 cells) can be obtained from, for example, the ATCC. Human umbilicalvein endothelial cells (HUVEC) can be isolated from human umbilicalveins as described. (Grant et al., “Matrigel induces thymosin h 4 genein differentiating endothelial cells”, J Cell Sci 1995; 108:3685-94.).HUVEC cells can be used as a positive control as a cell line thatexpress ATP synthase on the cell surface. Cells can be cultured in DMEM(Life Technologies, Carlsbad, Calif.) with 1% penicillin streptomycinand 10% serum replacement medium 3 (Sigma, St. Louis, Mo.) to minimizethe presence of plasminogen. Low-pH (6.7) medium can be prepared byreducing bicarbonate to 10 mmol/L at 5% CO2 and supplementing with 34mmol/L NaCl to maintain osmolality or incubation of 22 mmol/Lbicarbonate medium under 17% CO2 conditions. The method of lowering pHused can be varied by experimental constraints and assay.

Flow cytometry. To assure ATP synthase is functional on the cell surfaceof the tumor cell line, flow cytometry experiments can be performed. Forexample, A549 Cell lines can be cultured in varying pH medium (10, 22,and 44 mmol/L bicarbonate DMEM), under hypoxia (0.5% O2, 5% CO₂, N2balanced) versus normoxia (21% O2, 5% CO2) for 0, 12, 24, 48, and 72hours. Live cells can be blocked, incubated with anti-13-subunitantibody, washed, blocked, incubated with a secondary goat anti-rabbitantibody-FITC (Southern Biotech, Birmingham, Ala.), and again washed,with all steps performed at 4 degrees C. Propidium iodide (BDBiosciences, San Jose, Calif.) can be included with all samples todiscriminate cells with compromised membranes. The mean fluorescentintensity of FITC in 10,000 cells can be quantified by FACSCalibur flowcytometer (Becton Dickinson, Franklin Lakes, N.J.) and cells withpropidium iodide uptake can be excluded to eliminate detection ofmitochondrial ATP synthase on CELLQuest software (BD Biosciences).

Cell surface ATP generation assay. A549 or 1-LN cells (60,000 per well)in 96-well plates can be refreshed with medium and treated withangiostatin, angiosatain variant, anti-beta-subunit antibody, rabbit IgGraised to bovine serum albumin (Organon Teknika, West Chester, Pa.),piceatannol (a known inhibitor of ATP synthase F1 used as a positivecontrol, Sigma), or medium alone for 30 minutes at 37 degrees C., 5%CO2. Cells can be then incubated with 0.05 mmol/L ADP for 20 seconds.Supernatants can be removed and assayed for ATP production byCellTiterGlo luminescence assay (Promega, Madison, Wis.) as described(23). Cellysates can be similarly analyzed to confirm that intracellularpools of ATP did not vary under any conditions. Recordings can be madeon the Luminoskan Ascent (Thermo Labsystems, Helsinki, Finland). Dataare expressed in moles of ATP per cell based on standards determined foreach independent experiment.

Cell proliferation assay. The effect of angiostatin on cancer cell linescan be assessed with a3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt (MTS) proliferation assay in serum-free medium. Relative cellnumbers in each well of a 96-well microplate after incubation for 20hours, 37 degrees C., and 5% COz in the presence or absence ofangiostatin can be determined using the AQueous One Cell ProliferationAssay (Promega) per protocol of the manufacturer. Medium pH can beregulated at 5% CO₂ through bicarbonate concentration.

Assessment of cellular cytotoxicity. To quantify cell death and celllysis, the activity of lactate dehydrogenase (LDH) released from thecytosol into supernatant can be measured with the Cytotoxicity Detectionkit (Roche, Indianapolis, Ind.). Cancer cells (e.g. A549 cells) (5,000per well) treated with angiostatin, angiostatin variant,anti-beta-subunit antibody, rabbit IgG, cariporide, and Triton X (adetergent used to permeabilize cells as a positive control) can beincubated at 37 degrees C. and 5% COz or 17% CO2 for 15 hours at neutraland low pH conditions, respectively. An index of cytotoxicity can becalculated by dividing the average absorbance from treated samples inquadruplicate by the average absorbance from untreated samples inquadruplicate corresponding to the same pH medium. Assessment ofcellular necrosis and apoptosis. To determine the mode of angiostatininduced cell death a histone-DNA ELISA can be performed. The effects ofangiostatin, angiostatin variants, anti-beta-subunit antibody, rabbitIgG, and cariporide on A549 cells (5,000 per well) can be determinedusing an ELISA apoptosis and necrosis assay (Roche) that is dependent ondetection of extranuclear histone-DNA fragments. Apoptosis or necrosiscan be determined from, respectively, the cellysates or supernatants ofquadruplicate samples after 15 hours of incubation at 37 degrees C., inthe presence or absence of agents. The apoptotic or necrotic indices canbe calculated by dividing the average absorbance from treated samples inquadruplicate by the average absorbance from untreated samples inquadruplicate corresponding to the same pH medium. Medium pH can beregulated by incubation at 5% CO2 or 17% CO2.

Intracellular pH (pHi) measurement. pHi can be measured by fluorescencein cells plated on 35-mm microwell dishes with glass coverslips (MatTek,Ashland, Mass.). Cells can be plated on growth factor-reduced,phenol-red free Matrigel (BD Biosciences). After overnight growth,medium can be changed and cells can be loaded with the pH-sensitivefluorescent dye cSNARF (Molecular Probes, Eugene, Oreg.) for 15 minutesfollowed by 20 minutes recovery in fresh medium. Cells can then bemounted on a microscope stage at 37 degrees C., 5% CO2 for 1 hour-longcollection of emission spectra from which pHi can be calculated asdescribed from fields containing between 7 and 15 cells each (Wahl M L,Grant D S. “Effects of microenvironmental extracellular pH andextracellular matrix proteins on angiostatin's activity and onintracellular pH”, Gen Pharmacol 2002; 35:277-85). At the start ofspectra collection, medium can be removed from the dish and cells can bechallenged with 1 mL of fresh medium in the presence or absence of pHinhibitors angiostatin, anti-beta-subunit, rabbit IgG, or cariporide, asodium-proton exchange inhibitor. Medium pH can be regulated bybicarbonate concentration, as described above, with fixed % CO2.

1-18. (canceled)
 19. A method of preparing a conditionally activebiologic protein, the method comprising the steps of: i. selecting awild-type biologic protein selected from the group consisting ofenzymes, non-adrenergic neuropeptides, thrombolytics, renin, argininevasopressin, and angiostatin; ii. evolving the DNA which encodes thewild-type biologic protein using one or more evolutionary techniques tocreate mutant DNAs; iii. expressing the mutant DNAs to obtain at leastone mutant protein; iv. subjecting the at least one mutant protein andthe wild-type protein to an assay under a normal physiological conditionand to an assay under an aberrant condition; and v. selecting theconditionally active biologic protein from the at least one mutantprotein which exhibits both (a) a decrease in activity in the assay atthe normal physiological condition compared to the wild-type protein,and (b) an increase in activity in the assay under the aberrantcondition compared to the wild-type protein.
 20. The method of claim 19,wherein the biological protein is a therapeutic protein.
 21. The methodof claim 19, wherein the biological protein is naturally occurring. 22.The method of claim 19, wherein the biological protein is in a pro-form.23. The method of claim 19, wherein the evolving step comprising atechnique selected from the group consisting of non-stochasticpolynucleotide chimerization and non-stochastic site-directed pointmutagenesis.
 24. The method of claim 19, wherein the evolving stepcomprising a technique selected from the group consisting ofsubstituting one or more nucleotides for a different nucleotide,deleting one or more nucleotides, adding one or more nucleotides andcombinations thereof.
 25. The method of claim 19, wherein the evolvingstep comprising a technique selected from the group consisting of PCR,error-prone PCR, shuffling, oligonucleotide-directed mutagenesis,assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassettemutagenesis, recursive ensemble mutagenesis, exponential ensemblemutagenesis, site-specific mutagenesis, gene reassembly, gene sitesaturated mutagenesis, in vitro mutagenesis, ligase chain reaction,oligonucleotide synthesis and combination thereof.
 26. The method ofclaim 19, wherein the expressing step is performed in a host organism orcell selected from the group consisting of a gram negative bacterium, agram positive bacterium and a eukaryotic cell.
 27. The method of claim26, wherein the gram negative bacterium is selected from the groupconsisting of Escherichia coli and Pseudomonas fluorescens.
 28. Themethod of claim 26, wherein the gram positive bacterium is selected fromthe group consisting of Streptomyces diversa, Lactobacillus gasseri,Lactococcus lactis, Lactococcus cremoris, and Bacillus subtilis.
 29. Themethod of claim 26, wherein the eukaryotic organism is selected from thegroup consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe,Pichia pastoris, Kluyveromyces lactis, Hansenula plymorpha, orAspergillus niger.
 30. The method of claim 26, wherein the eukaryoticcell is selected from the group consisting of a Drosophila S2 cell, aSpodoptera Sf9 cell, a Bowes melanoma, and a plant cell.
 31. The methodof claim 26, wherein the eukaryotic cell is selected from the groupconsisting of a COS-7 cell line, a C127 cell line, a 3T3 cell line, aCHO cell line, a HeLa cell line and a BHK cell line.
 32. The method ofclaim 19, wherein the expressing step employs an inducing agent toinduce biological protein expression.
 33. The method of claim 19,wherein the normal physiological condition is selected from one or moreof normal physiological temperature, pH, osmotic pressure, osmolality,oxidation and electrolyte concentration.
 34. The method of claim 19,wherein the normal physiological condition is temperature; and whereinthe conditionally active biologic protein is inactive at the normalphysiological temperature, and is active at an aberrant temperature lessthan the normal physiological temperature.
 35. A conditionally activebiologic protein prepared by the method of claim 19, wherein the proteinis reversibly or irreversibly inactivated at the normal physiologicalcondition.
 36. The conditionally active biologic protein of claim 35,wherein the protein is reversibly inactivated at the wild type normalphysiological conditions.